Use of hla genetic status to assess or select treatment of celiac disease

ABSTRACT

Provided herein are methods of adjusting or selecting a gluten peptide therapy based on the human leukocyte antigen (HLA) genotype, in particular HLA-DQ2.5 homozygosity, of a subject having or suspected of having Celiac disease. Also provided herein are methods of identifying (e.g., diagnosing) a subject, such as a subject having or suspected of having Celiac disease and/or assessing the efficacy of treatment of Celiac disease, e.g. by determining responsiveness to a therapeutic gluten peptide composition or cytokine response, and kits relating thereto.

RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. § 119(e) of U.S.provisional application No. 62/057,167, filed Sep. 29, 2014, the entirecontents of which is incorporated by reference herein in their entirety.

BACKGROUND OF THE INVENTION

Celiac disease, also known as coeliac disease or Celiac sprue (Coeliacsprue), affects approximately 1% of people in Europe and North America.In many of those affected, Celiac disease is unrecognised, but thisclinical oversight is now being rectified with greater clinicalawareness. A gluten free diet is the only currently approved treatmentfor Celiac disease, and because regular ingestion of as little as 50 mgof gluten (equivalent to 1/100^(th) of a standard slice of bread) candamage the small intestine; chronic inflammation of the small bowel iscommonplace in subjects on a gluten free diet. Persistent inflammationof the small intestine has been shown to increase the risk of cancer,osteoporosis and death. As gluten is so widely used, for example, incommercial soups, sauces, ice-creams, etc., maintaining a gluten-freediet is difficult.

Celiac disease occurs in genetically susceptible individuals who possesseither HLA-DQ2.5 (encoded by the genes HLA-DQA1*05 and HLA-DQB1*02)accounting for about 90% of individuals, HLA-DQ2.2 (encoded by the genesHLA-DQA1*02 and HLA-DQB1*02), or HLA-DQ8 (encoded by the genesHLA-DQA1*03 and HLA-DQB1*0302). Without wishing to be bound by theory,it is believed that such individuals mount an inappropriate HLA-DQ2-and/or DQ8-restricted CD4⁺ T cell-mediated immune response to peptidesderived from the aqueous-insoluble proteins of wheat flour, gluten, andrelated proteins in rye and barley.

SUMMARY OF THE INVENTION

As described herein, it has been found that subjects having Celiacdisease that are homozygous for HLA-DQ2.5 have higher levels ofcirculating cytokines and/or adverse symptoms after administration of agluten peptide composition. Accordingly, aspects of the disclosurerelate to methods of selecting or adjusting a gluten peptide treatmentbased on the human leukocyte antigen (HLA) genotype of a subject beingtreated, such as a subject having or suspected of having Celiac disease

Accordingly, aspects of the disclosure relate to a method of selectingor adjusting a gluten peptide treatment for a subject that has or issuspected of having Celiac disease based on a human leukocyte antigen(HLA) genotype of the subject.

In some embodiments of any one of the methods provided, the methodfurther comprises assessing the HLA genotype of the subject.

In some embodiments of any one of the methods provided, assessingcomprises determining the sequence of each copy of an HLA-DQA gene andeach copy of an HLA-DQB gene in the subject. In some embodiments of anyone of the methods provided, determining comprises performing anucleic-acid based assay on each copy of the HLA-DQA gene, or a portionthereof, and each copy of the HLA-DQB gene, or a portion thereof. Insome embodiments of any one of the methods provided, the nucleic-acidbased assay is a probe-based assay or a sequencing assay.

In some embodiments of any one of the methods provided, assessingfurther comprises identifying the subject as having a homozygousHLA-DQ2.5 genotype or a non-homozygous HLA-DQ2.5 genotype. In someembodiments of any one of the methods provided, the non-homozygousHLA-DQ2.5 genotype is a heterozygous HLA-DQ2.5 genotype. In someembodiments of any one of the methods provided, the heterozygousHLA-DQ2.5 genotype is HLA-DQ^(25/22), HLA-DQ^(25/7), or HLA-DQ^(25/8).

In some embodiments of any one of the methods provided, the methodfurther comprises decreasing a dose of the gluten peptide treatment ifthe subject has a homozygous HLA-DQ2.5 genotype; or maintaining orincreasing the dose of the gluten peptide treatment if the subject has anon-homozygous HLA-DQ2.5 genotype.

In some embodiments of any one of the methods provided, any one of thegluten peptide compositions may comprise at least one peptide comprisingat least one amino acid sequence selected from PFPQPELPY (SEQ ID NO: 1),PQPELPYPQ (SEQ ID NO:2), PFPQPEQPF (SEQ ID NO:3), PQPEQPFPW (SEQ IDNO:4), PIPEQPQPY (SEQ ID NO:5) and EQPIPEQPQ (SEQ ID NO:6).

In some embodiments of any one of the methods provided, any one of thegluten peptide compositions may comprise:

-   -   a) a first peptide comprising the amino acid sequence PFPQPELPY        (SEQ ID NO: 1) and the amino acid sequence PQPELPYPQ (SEQ ID        NO:2);    -   b) a second peptide comprising the amino acid sequence PFPQPEQPF        (SEQ ID NO:3) and the amino acid sequence PQPEQPFPW (SEQ ID        NO:4); and    -   c) a third peptide comprising the amino acid sequence EQPIPEQPQ        (SEQ ID NO:6) and the amino acid sequence PIPEQPQPY (SEQ ID        NO:5).

In some embodiments of any one of the methods provided, any one of thegluten peptide compositions may comprise a first peptide that comprisesthe amino acid sequence LQPFPQPELPYPQPQ; a second peptide that comprisesthe amino acid sequence QPFPQPEQPFPWQP (SEQ ID NO: 7); and a thirdpeptide that comprises the amino acid sequence PEQPIPEQPQPYPQQ (SEQ IDNO: 8). In some embodiments of any one of the methods provided, any oneof the gluten peptide compositions may comprise a first peptide thatcomprises the amino acid sequence ELQPFPQPELPYPQPQ (SEQ ID NO: 9),wherein the N-terminal glutamate is a pyroglutamate and the C-terminalglutamine is amidated; a second peptide that comprises the amino acidsequence EQPFPQPEQPFPWQP (SEQ ID NO: 10), wherein the N-terminalglutamate is a pyroglutamate and the C-terminal proline is amidated; anda third peptide that comprises the amino acid sequence EPEQPIPEQPQPYPQQ(SEQ ID NO: 11), wherein the N-terminal glutamate is a pyroglutamate andthe C-terminal glutamine is amidated.

In some embodiments of any one of the methods provided, the dose is oris decreased to less than 300 micrograms if the subject has a homozygousHLA-DQ2.5 genotype. In some embodiments of any one of the methodsprovided, the dose is or is decreased to less than 150 micrograms if thesubject has a homozygous HLA-DQ2.5 genotype. In some embodiments of anyone of the methods provided, the dose is selected to be up to 300micrograms if the subject has a heterozygous HLA-DQ2.5 genotype. In someembodiments of any one of the methods provided herein, the amountselected based on HLA-DQ2.5 genotype is any one of the foregoing.

Other aspects of the disclosure relate to a method of measuring a levelof at least one circulating cytokine or chemokine in a subject that hasor is suspected of having Celiac disease, wherein the subject has beenadministered a first composition comprising at least one gluten peptidein an amount selected based on a human leukocyte antigen (HLA) genotypeof the subject, and assessing the likelihood the subject has Celiacdisease.

In some embodiments of any one of the methods provided, the methodfurther includes comprises assessing the HLA genotype of the subject.

In some embodiments of any one of the methods provided assessingcomprises determining the sequence of each copy of an HLA-DQA gene andeach copy of an HLA-DQB gene in the subject. In some embodiments of anyone of the methods provided, determining comprises performing anucleic-acid based assay on each copy of the HLA-DQA gene, or a portionthereof, and each copy of the HLA-DQB gene, or a portion thereof. Insome embodiments of any one of the methods provided, the nucleic-acidbased assay is a probe-based assay or a sequencing assay.

In some embodiments of any one of the methods provided, assessingfurther comprises identifying the subject as having a homozygousHLA-DQ2.5 genotype or a non-homozygous HLA-DQ2.5 genotype. In someembodiments of any one of the methods provided, the non-homozygousHLA-DQ2.5 genotype is a heterozygous HLA-DQ2.5 genotype. In someembodiments of any one of the methods provided, the heterozygousHLA-DQ2.5 genotype is HLA-DQ²⁵¹²², HLA-DQ²⁵¹⁷, or HLA-DQ²⁵¹⁸.

In some embodiments of any one of the methods provided, the methodfurther comprises decreasing the amount of a composition, e.g., a firstcomposition, a second composition, or both, comprising at least onegluten peptide administered to the subject if the subject has ahomozygous HLA-DQ2.5 genotype; or maintaining or increasing the amountof a composition, e.g., a first composition, a second composition, orboth, comprising at least one gluten peptide administered to the subjectif the subject has a non-homozygous HLA-DQ2.5 genotype. In someembodiments of any one of the methods provided, the methods furthercomprises selecting any one of the amounts or doses as described herein.

In some embodiments of any one of the methods provided, a composition,e.g., a first composition and/or a second composition, comprising atleast one gluten peptide comprises: a first peptide comprising the aminoacid sequence PFPQPELPY (SEQ ID NO: 1) and the amino acid sequencePQPELPYPQ (SEQ ID NO:2);

a second peptide comprising the amino acid sequence PFPQPEQPF (SEQ IDNO:3) and the amino acid sequence PQPEQPFPW (SEQ ID NO:4); and

a third peptide comprising the amino acid sequence EQPIPEQPQ (SEQ IDNO:6) and the amino acid sequence PIPEQPQPY (SEQ ID NO:5).

In some embodiments of any one of the methods provided, the firstpeptide comprises the amino acid sequence LQPFPQPELPYPQPQ (SEQ ID NO:62); the second peptide comprises the amino acid sequence QPFPQPEQPFPWQP(SEQ ID NO: 7); and the third peptide comprises the amino acid sequencePEQPIPEQPQPYPQQ (SEQ ID NO: 8).

In some embodiments of any one of the methods provided, the firstpeptide comprises the amino acid sequence ELQPFPQPELPYPQPQ (SEQ ID NO:9), wherein the N-terminal glutamate is a pyroglutamate and theC-terminal glutamine is amidated; the second peptide comprises the aminoacid sequence EQPFPQPEQPFPWQP (SEQ ID NO: 10), wherein the N-terminalglutamate is a pyroglutamate and the C-terminal proline is amidated; andthe third peptide comprises the amino acid sequence EPEQPIPEQPQPYPQQ(SEQ ID NO: 11), wherein the N-terminal glutamate is a pyroglutamate andthe C-terminal glutamine is amidated.

In some embodiments of any one of the methods provided, the amount ofthe composition comprising at least one gluten peptide is or isdecreased to less than 300 micrograms if the subject has a homozygousHLA-DQ2.5 genotype. In some embodiments of any one of the methodsprovided the amount of the composition comprising at least one glutenpeptide is or is decreased to less than 150 micrograms if the subjecthas a homozygous HLA-DQ2.5 genotype.

In some embodiments of any one of the methods provided, the methodfurther comprises obtaining a sample from the subject and the measuringis performed on the sample.

In some embodiments of any one of the methods provided, the sample fromthe subject is obtained 1 hour to 6 hours after the subject has beenadministered the first composition. In some embodiments of any one ofthe methods provided, the sample from the subject is obtained 4 hours to6 hours after the subject has been administered the first composition.In some embodiments of any one of the methods provided, the sample fromthe subject is a plasma or serum sample.

In some embodiments of any one of the methods provided, the subject hasbeen administered the first composition by injection. In someembodiments of any one of the methods provided, the subject has beenadministered the first composition by oral administration.

In some embodiments of any one of the methods provided, the methodfurther comprises administering the first composition to the subjectprior to measuring the level of the at least one circulating cytokine orchemokine. In some embodiments of any one of the methods provided, theat least one circulating cytokine or chemokine is MCP-1, IP-10, IL-6,IL-8, G-CSF, IL-2, IL-IRA, GRO, EOTAXIN, GM-CSF, IL-10, TNFa, IFNa2,MIP-1b, IL-12P70, IL-la, IL-17A, EGF, MIP-la, FRACTALKINE, IFNg, VEGF,IL-9, FGF-2, IL-lb, Flt-3L, I-15, TNFb, IL-12(P40), MCP-3, IL-4, MDC,IL-13, TGF-a, IL-3, IL-5, IL-7 or sCD40L.

In some embodiments of any one of the methods provided, an elevatedlevel of the at least one circulating cytokine or chemokine compared toa control level of the at least one circulating cytokine or chemokineindicates that the subject has Celiac disease, and the step of assessingcomprises comparing the level of the at least one circulating cytokineor chemokine to a control level of the at least one circulating cytokineor chemokine. In some embodiments of any one of the methods provided,the control level is a baseline level. In some embodiments of any one ofthe methods provided, the baseline level is a level of the at least onecirculating cytokine or chemokine prior to administration of the firstcomposition.

In some embodiments of any one of the methods provided, the methodfurther comprises recording whether or not the subject has celiacdisease based on the assessing.

In some embodiments of any one of the methods provided, the methodfurther comprises treating, suggesting a treatment, or givinginformation in regard to a treatment to the subject.

In some embodiments of any one of the methods provided, the methodfurther comprises: decreasing a dose of the gluten peptide treatment ifthe subject has a homozygous HLA-DQ2.5 genotype; or maintaining orincreasing the dose of the gluten peptide treatment if the subject has anon-homozygous HLA-DQ2.5 genotype

In some embodiments of any one of the methods provided, measuring thelevel of the at least one circulating cytokine or chemokine comprises animmuno-based assay. In some embodiments of any one of the methodsprovided, the immuno-based assay comprises an ELISA or a multiplexbead-based assay.

In some embodiments of any one of the methods provided, the methodfurther comprises measuring a T cell response to the first compositioncomprising the at least one gluten peptide.

Other aspects of the disclosure relate to a method for assessingtolerance to a gluten peptide in a subject having Celiac disease, themethod comprising: measuring a level of at least one circulatingcytokine or chemokine in a subject having Celiac disease, wherein thesubject has been administered a first composition comprising at leastone gluten peptide in an amount selected based on a human leukocyteantigen (HLA) genotype of the subject, and assessing the tolerance ofthe subject to the at least one gluten peptide based on the measuring.

In some embodiments of any one of the methods provided, the methodfurther comprises assessing the HLA genotype of the subject. In someembodiments of any one of the methods provided, assessing comprisesdetermining the sequence of each copy of an HLA-DQA gene and each copyof an HLA-DQB gene in the subject. In some embodiments of any one of themethods provided, determining comprises performing a nucleic-acid basedassay on each copy of the HLA-DQA gene, or a portion thereof, and eachcopy of the HLA-DQB gene, or a portion thereof. In some embodiments ofany one of the methods provided the nucleic-acid based assay is aprobe-based assay or a sequencing assay.

In some embodiments of any one of the methods provided, assessingfurther comprises identifying the subject as having a homozygousHLA-DQ2.5 genotype or a non-homozygous HLA-DQ2.5 genotype. In someembodiments of any one of the methods provided, the non-homozygousHLA-DQ2.5 genotype is a heterozygous HLA-DQ2.5 genotype. In someembodiments of any one of the methods provided, the heterozygousHLA-DQ2.5 genotype is HLA-DQ²⁵¹²², HLA-DQ²⁵¹⁷, or HLA-DQ²⁵¹⁸.

In some embodiments of any one of the methods provided, the methodfurther comprises: decreasing the amount of the first compositioncomprising at least one gluten peptide administered to the subject ifthe subject has a homozygous HLA-DQ2.5 genotype; or maintaining orincreasing the amount of the first composition comprising at least onegluten peptide administered to the subject if the subject has anon-homozygous HLA-DQ2.5 genotype.

In some embodiments of any one of the methods provided, a composition,e.g., a first composition and/or a second composition, comprising atleast one gluten peptide comprises:

a first peptide comprising the amino acid sequence PFPQPELPY (SEQ IDNO: 1) and the amino acid sequence PQPELPYPQ (SEQ ID NO:2);

a second peptide comprising the amino acid sequence PFPQPEQPF (SEQ IDNO:3) and the amino acid sequence PQPEQPFPW (SEQ ID NO:4); and

a third peptide comprising the amino acid sequence EQPIPEQPQ (SEQ IDNO:6) and the amino acid sequence PIPEQPQPY (SEQ ID NO:5).

In some embodiments of any one of the methods provided, the firstpeptide comprises the amino acid sequence LQPFPQPELPYPQPQ (SEQ ID NO:62); the second peptide comprises the amino acid sequence QPFPQPEQPFPWQP(SEQ ID NO: 7); and the third peptide comprises the amino acid sequencePEQPIPEQPQPYPQQ (SEQ ID NO: 8).

In some embodiments of any one of the methods provided, the firstpeptide comprises the amino acid sequence ELQPFPQPELPYPQPQ (SEQ ID NO:9), wherein the N-terminal glutamate is a pyroglutamate and theC-terminal glutamine is amidated; the second peptide comprises the aminoacid sequence EQPFPQPEQPFPWQP (SEQ ID NO: 10), wherein the N-terminalglutamate is a pyroglutamate and the C-terminal proline is amidated; andthe third peptide comprises the amino acid sequence EPEQPIPEQPQPYPQQ(SEQ ID NO: 11), wherein the N-terminal glutamate is a pyroglutamate andthe C-terminal glutamine is amidated.

In some embodiments of any one of the methods provided, the amount ofthe composition comprising at least one gluten peptide is or isdecreased to less than 300 micrograms if the subject has a homozygousHLA-DQ2.5 genotype. In some embodiments of any one of the methodsprovided, the amount of the composition comprising at least one glutenpeptide is or is decreased to less than 150 micrograms if the subjecthas a homozygous HLA-DQ2.5 genotype.

In some embodiments of any one of the methods provided, the methodfurther comprises obtaining a sample from the subject and the measuringis performed on the sample. In some embodiments of any one of themethods provided, the sample from the subject is obtained 1 hour to 6hours after the subject has been administered the first composition. Insome embodiments of any one of the methods provided, the sample from thesubject is obtained 4 hours to 6 hours after the subject has beenadministered the first composition. In some embodiments of any one ofthe methods provided, the sample from the subject is a plasma or serumsample.

In some embodiments of any one of the methods provided, the subject hasbeen administered the first composition by injection. In someembodiments of any one of the methods provided, the subject has beenadministered the first composition by oral administration.

In some embodiments of any one of the methods provided, the methodfurther comprises administering the first composition to the subjectprior to measuring the level of the at least one circulating cytokine orchemokine. In some embodiments of any one of the methods provided, theat least one circulating cytokine or chemokine is MCP-1, IP-10, IL-6,IL-8, G-CSF, IL-2, IL-IRA, GRO, EOTAXIN, GM-CSF, IL-10, TNFa, IFNa2,MIP-lb, IL-12P70, IL-la, IL-17A, EGF, MIP-la, FRACTALKINE, IFNg, VEGF,IL-9, FGF-2, IL-lb, Flt-3L, I-15, TNFb, IL-12(P40), MCP-3, IL-4, MDC,IL-13, TGF-a, IL-3, IL-5, IL-7 or sCD40L.

In some embodiments of any one of the methods provided, an elevatedlevel of the at least one circulating cytokine or chemokine compared toa control level of the at least one circulating cytokine or chemokineindicates that the subject has Celiac disease, and the step of assessingcomprises comparing the level of the at least one circulating cytokineor chemokine to a control level of the at least one circulating cytokineor chemokine. In some embodiments of any one of the methods provided,the control level is a baseline level. In some embodiments of any one ofthe methods provided, the baseline level is a level of the at least onecirculating cytokine or chemokine prior to administration of the firstcomposition.

In some embodiments of any one of the methods provided, the methodfurther comprises recording whether or not the subject has celiacdisease based on the assessing. In some embodiments of any one of themethods provided, the method further comprises treating, suggesting atreatment, or giving information in regard to a treatment to thesubject.

In some embodiments of any one of the methods provided, the methodfurther comprises: decreasing a dose of the gluten peptide treatment ifthe subject has a homozygous HLA-DQ2.5 genotype; or maintaining orincreasing the dose of the gluten peptide treatment if the subject has anon-homozygous HLA-DQ2.5 genotype

In some embodiments of any one of the methods provided, measuring thelevel of the at least one circulating cytokine or chemokine comprises animmuno-based assay. In some embodiments of any one of the methodsprovided, the immuno-based assay comprises an ELISA or a multiplexbead-based assay.

In some embodiments of any one of the methods provided, the methodfurther comprises measuring a T cell response to the first compositioncomprising the at least one gluten peptide.

Other aspects of the disclosure relate to a kit comprising i) the firstcomposition as defined by any one of the methods provided, and ii) abinding partner for the at least one cytokine or chemokine as defined byany one of the methods provided. In some embodiments of any one of themethods provided, the kit further comprises iii) a means for injectingthe first composition.

Other aspects of the disclosure relate to a method comprising:administering to a subject that has or is suspected of having Celiacdisease a first composition comprising at least one gluten peptide in anamount selected based on an HLA genotype of the subject, measuring a Tcell response to a second composition comprising at least one glutenpeptide in a sample from the subject, and assessing the likelihood thatthe subject has Celiac disease.

In some embodiments of any one of the methods provided, the firstcomposition and the second composition comprise the same gluten peptideor peptides. In some embodiments of any of the methods provided, thesample is contacted with the second composition.

In some embodiments of any one of the methods provided, the methodfurther comprises obtaining the sample from the subject.

In some embodiments of any one of the methods provided, the the subjectis orally administered or directed to consume gluten for at least threedays.

In some embodiments of any one of the methods provided, the themeasuring step is performed six days after the last of the gluten isorally administered or consumed.

In some embodiments of any one of the methods provided, IFN-gamma ismeasured. In some embodiments of any one of the methods provided, IP-10is measured.

The details of one or more embodiments of the disclosure are set forthin the description below. Other features or advantages of the presentdisclosure will be apparent from the following drawings and detaileddescription of several embodiments, and also from the appending claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentdisclosure, which can be better understood by reference to one or moreof these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1 is a table showing the number of subjects in each of Cohorts 1,2, and 7 that were homozygous for HLA-DQ2.5, heterozygous HLA-DQ2.5/2.2or 2.5/7, heterozygous HLA-DQ2.5 or HLA-DQ2.5/unknown.

FIG. 2 is a table showing the median peak fold change in circulatingcytokines from pre-dose after 1^(st) dose of the gluten peptidecomposition. Other means non-HLA-DQ2.5 homozygous subjects. Sx=symptoms.

FIG. 3 is a graph showing the peak fold change in concentration of IL-2at 4-6 hours after dose of the gluten peptide composition in subjectsthat were homozygous for HLA-DQ2.5 (n=22) or heterozygous HLA-DQ2.5/2.2or 2.5/7, heterozygous HLA-DQ2.5 or HLA-DQ2.5/unknown (n=60).

FIG. 4 is a graph showing peak fold change in concentration of IL-2,IL-8, IL-10, TNF-α, MIP-1β, GM-CSF, Eotaxin, IP-10, and MCP-1 at 4-6hours after dose of the gluten peptide composition in subjects that werehomozygous for HLA-DQ2.5 (n=22) or heterozygous HLA-DQ2.5/2.2 or 2.5/7,heterozygous HLA-DQ2.5 or HLA-DQ2.5/unknown (n=60).

DETAILED DESCRIPTION OF THE INVENTION

As described herein, it has been found that subjects who are homozygousfor the HLA-DQ2.5 genotype have higher levels of circulating cytokinesand/or adverse events in response to administration of a gluten peptidetreatment. Accordingly, aspects of the disclosure relate to selecting oradjusting a gluten peptide treatment for a subject (e.g., a subjecthaving or suspected of having Celiac disease) based on the humanleukocyte antigen (HLA) genotype of the subject.

Methods

Aspects of the disclosure relate to a method, comprising selecting oradjusting a gluten peptide treatment for a subject that has or issuspected of having Celiac disease based on a human leukocyte antigen(HLA) genotype of the subject. Gluten peptide treatments are furtherdescribed herein.

Another aspect of the disclosure relates to a method of identifying(e.g., diagnosing) a subject, such as a subject having or suspected ofhaving Celiac disease based on the HLA-DQ2.5 genotype.

Yet another aspect of the disclosure relates to a method of assessingthe efficacy of treatment of Celiac disease (e.g., responsiveness to atherapeutic gluten peptide composition) based on the HLA-DQ2.5 genotype

In some embodiments, the method further comprises assessing the HLAgenotype of the subject. In some embodiments of any one of the methodsdescribed herein, the assessing comprises determining the sequence ofeach copy of an HLA-DQA gene and each copy of an HLA-DQB gene (includingdetermining the sequence of a portion thereof) in the subject. HLA genesand genotypes are further described herein.

In some embodiments of any one of the methods described herein,determining the sequence comprises reading sequence information, e.g.,on a chart, on a print-out, or on a computer such as in a database ofsequence information. In some embodiments, the sequence information maybe conveyed as a symbol or other words, numbers or letters that indicatethe sequence (e.g., DQA1*05 or DQB1*02).

In some embodiments, assessing the genotype includes being given orbeing told the genotype information for the subject. In someembodiments, assessing the genotype of subject includes obtaininggenotype information for the subject from an individual, e.g., a patientprovider or laboratory personnel, a tangible medium, e.g., on a chart orprint out, or an intangible medium, e.g., a database.

In some embodiments, assessing the genotype includes performing an assayon a patient or patient sample.

In some embodiments of any one of the methods described herein,determining comprises performing a nucleic-acid based assay on one orboth copies of the HLA-DQA gene, or a portion thereof, and on one orboth copies of the HLA-DQB gene, or a portion thereof. In someembodiments, the nucleic-acid based assay is a probe-based assay or asequencing assay. Nucleic-acid based assays are further describedherein.

In some embodiments of any one of the methods described herein,assessing further comprises identifying the subject as having ahomozygous HLA-DQ2.5 genotype or a non-homozygous HLA-DQ2.5 genotype. Insome embodiments, the non-homozygous HLA-DQ2.5 genotype is aheterozygous HLA-DQ2.5 genotype. HLA-DQ2.5 genotypes are furtherdescribed herein.

In some embodiments of any one of the methods described herein, themethod further comprises decreasing a dose of the gluten peptidetreatment if the subject has a homozygous HLA-DQ2.5 genotype; ormaintaining or increasing the dose of the gluten peptide treatment ifthe subject has a non-homozygous HLA-DQ2.5 genotype.

In some embodiments of any one of the methods described herein, themethod further comprises measuring a level of at least one circulatingcytokine or chemokine in a subject that has or is suspected of havingceliac disease, wherein the subject has been administered a compositioncomprising a decreased dose of at least one gluten peptide as describedherein based on the HLA-DQ2.5 genotype.

In some embodiments of any one of the methods provided, the dose is oris decreased to less than 300 micrograms of the gluten peptides if thesubject has a homozygous HLA-DQ2.5 genotype. In some embodiments of anyone of the methods provided, the dose is or is decreased to less than150 micrograms if the subject has a homozygous HLA-DQ2.5 genotype. Insome embodiments of any one of the methods provided, the dose is or isincreased to up to 300 micrograms if the subject has a heterozygousHLA-DQ2.5 genotype.

In some embodiments of any one of the methods provided, the dose of thegluten peptides is selected to be up to 300 micrograms if the subjecthas a heterozygous HLA-DQ2.5 genotype.

In some embodiments, the selected dose for a subject having a homozygousDQ2.5 genotype is less than the dose that would be selected for asubject having a heterozygous DQ2.5 genotype. In some embodiments, theselected dose for a subject having a heterozygous DQ2.5 genotype is morethan the dose that would be selected for a subject having a homozygousDQ2.5 genotype.

HLA Genotypes

Aspects of the disclosure relate to the assessment and/or assaying of anHLA genotype in a subject. In some embodiments, the assessing comprisesdetermining the presence or absence of one or more HLA-DQA alleles andone or more HLA-DQB alleles. In some embodiments, the assessingcomprises determining the sequence of one or more copies of an HLA-DQAgene and/or one or more copies of an HLA-DQB gene (including determiningthe sequence of a portion of each gene or determining the identity of aSNP associated with a particular allele) in a subject. In someembodiments, the HLA genotype is a homozygous HLA-DQ2.5 genotype or anon-homozygous HLA-DQ2.5 genotype. The non-homozygous HLA-DQ2.5 genotypemay be, e.g., a heterozygous HLA-DQ2.5 genotype. Exemplary heterozygousHLA-DQ2.5 genotypes include, but are not limited to, HLA-DQ2.5/2.2,HLA-DQ2.5/7, or HLA-DQ2.5/8.

Exemplary HLA-DQA and HLA-DQB alleles for the HLA-DQA and HLA-DQB genesare: HLA-DQ2.5 (DQA1*05 and DQB1*02), DQ2.2 (DQA1*02 and DQB1*02), DQ7(DQA1*05 and DQB1*0301) and DQ8 (DQA1*03 and DQB1*0302). Exemplarysequences for DQA and DQB alleles are shown below.

HLA-DQA1*0501 (Genbank accession number: AF515813.1)DQA1*0501 allele, 3′ UTR and partial cds (SEQ ID NO: 82)GGCCTGCGTTCAGTTGGTGCTTCCAGACACCAAGGGCCCTTGTGAATCCCATCCTGGAATGGAAGGTAAGATTGAGATTTGTTAGAGCTGAATCCGCAGTATGAGAGGAAGGAAAGTGGAGGAGGCTGTGGACATGAATGGTTGAAAGTTGTAGGGGAATTGGGAAGTGGCATGATGATGACATAGGAGCGGCCTAGGACCCATCCATCTCATGTCTGTCCTGTTGCAGGTGCATCGCCATCTACAGGAGCAGAAGAGTGGACTTGCTACATGACCTAGCATTATTTTCTGGCCCCATTTATCATATCCCTTTTCTCCTCCAAATGTTTCTCCTCTCACCTCTTCTGTGGGACTTAAATTGCTATATCTGCTCAGAGCTCACAAATGCCTTTGAATTATTTCCCTGACTTCCTGATTTTTTTCTTCTTAAGTGTTACCTACTAAGAGTTGCCTGGAGTAAGCCACCCAGCTACCTAATTCCTCAGTAACCTCCATCTATAATCTCCATGGAAGCAACAAATTCCCTTTATGAGATATATGTCAAATTTTTCCATCTTTCATCCAGGGCTGACTGAAACCGTGGCTAAGAATTGGGAGACTCTCTTGTTTCAAGCCAATTTAACATCATTTACCAGATCATTTGTCATGTCCAGTAACACAGAAGCAACCAACTACAGTATAGCCTGATAACATGTTGATTTCTTAGCTGACATTAATATTTCTTTCTTCCTTGTGTTCCCACCCTTGGCATTGCCACCCACCCCTCAATTAAGGCAACAATGAAGTTAATGGATACCCTCTGCCTTTGGCTCAGAAATGTTATAGCAAAAATTTTAAAATAAAAAAGTAAGTCTGTACTAATTTCAATATGACTTTTAAAAGTATGACAGAGAAATGGGTTGGGATAAAGGAAATTTGAATCTCAAAAATATCAATAGTGAAAAGTTATTCTCAAAACTTTAAATTTGTGAAGAATGATGACAGTAGAAGCCTTCCTCTCCCCTCCTCACCCTGAAGGAATAAAATTTCCTTAGGCAGGAAAAGAAATGGAAGTCAGAAAAACATTAGAATAAGACAATAATGTGGGTATCTGAAAAGGAACAAATACTCATTCCTCACATAGGGTTAGTGACAATGG HLA-DQA1*0505 (Genbank accession number: AH013295.2)HLA-DQA1*0505 allele, partial cds (SEQ ID NO: 83)CCAGTCCTGAGAGGAAAGAAAATACAATCAGTTTGTTATTAACTGAGGAAAGAATTAAGTGAAAGATGAATCTTAGGAAGCAGAAGGAAGTAAACCTAATCTCTGACTAAGAAAGCTAAATACCATAATAACTCATTCATTCCTTCTTTTGTTTAATTACATTATTTAATCATAAGTCCGTGATGTGCCAGGCACTCAGGAAATAGTAAAAACTGGACATGTGATATTCTGCCCTTGTGTAGCGCACATTATAGTGGGAAAGAAAGCGCAATTTTAACCGGACAACTACCAACAATAAGAGCGGAGGAAGCAGGGGTTGGAAATGTCCACAGGCTGTGCCAAAGATGAAGCCCGTAATATTTGAAAGTCAGTTTCTTTCATCATTTTGTGTATTAAGGTTCTTTCTTCCCCTGTTCTCCACCTTCCTGCTTGTCATCTTCACTCATCAGCTGACCACGTCGCCTCTTATGGTGTAAACTTGTACCAGTCTTACGGTCCCTCTGGCCAGTACACCCATGAATTTGATGGAGATGAGCAGTTCTACGTGGACCTGGGGAGGAAGGAGACTGTCTGGTGTTTGCCTGTTCTCAGACAATTTAGATTTGACCCGCAATTTGCACTGACAAACATCGCTGTCCTAAAACATAACTTGAACAGTCTGATTAAACGCTCCAACTCTACCGCTGCTACCAATGGTATGTGTCAACAATTCTGCCCCTCTTTACTGATTTATCCCTTCATACCAAGTTTCATTATTTTATTTCCAAGAGGTCCCCAGATCTTCTCATGGCAATTGCTGAAATTTTATCATCTCCCATCTCTAAAATCACATATTCCCATGTAATACAAGGGTCTTTCCATTATCCATTCATTAAATCCTTCTCGGAGAGGTCTCATCAACCTCCTACTTTATTAAACATGCCCACAGAGAGAAGGGCACAGGAATAAAGCGGAGGCAATGTGTCGTTGCTCCCAAGCAGAAGGTAAATAAGACCTCTTTGACTATCAGGTGGTGAAATGCTGGTAGGAGGGCTCTTCCAGGATGTAATGCAGAAGCTCATGGCAGAGCTATTCACACTTCACATCAGTGCTGTTTCCTCACCACAGAGGTTCCTGAGGTCACAGTGTTTTCCAAGTCTCCCGTGACACTGGGTCAGCCCAACATCCTCATCTGTCTTGTGGACAACATCTTTCCTCCTGTGGTCAACATCACATGGCTGAGCAATGGGCACTCAGTCACAGAAGGTGTTTCTGAGACCAGCTTCCTCTCCAAGAGTGATCATTCCTTCTTCAAGATCAGTTACCTCACCCTCCTCCCTTCTGCTGAGGAGAGTTATGACTGCAAGGTGGAGCACTGGGGACTGGACAAGCCTCTTCTGAAACACTGGGGTAAGGATGAGTTNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNTTTCTTTCTTTCTTTCTTTCTTTCTTTCTTTCTTTCTTTCTTTCTTTCTTTCTTTCTTTCTTTCTTTCTTTTTTGAAAGAATAAAGCAACAAAAGCAAAGATTTATTGAAAATGAAAGTACACTTCACATGGTGGGAGCGGGCCTGAGCATAGGGGCTCAAGAGCCACTTCATGGGTTTCTAATGATAGACTTCACTCTCCTCCCTAAGCTGGGGACCATGAGTCTTTGCAGAGCCAACCCTCCACCCCATCCCATCCCACACACATGCACATGAGCACACTCTGCTTTCTGACCTCAACGACTTCATATCCACAGAGCCTGAGATTCCAGCCCCTATGTCAGAGCTCACAGAGACTGTGGTCTGCGCCCTGGGGTTGTCTGTGGGCCTCGTGGGCATTGTGGTGGGCACTGTCTTCATCATCCGAGGCCTGCGTTCAGTTGGTGCTTCCAGACACCAAGGGCCCTTGTGAATCCCATCCTGGAATGGAAGGTAAGATTGAGATTTGTTAGAGCTGAATCCGCAGTATGAGAGGAAGGAAAGTGGAGGAGGCTGTGGACATGAATGGTTGAAAGTTGTAGGGGAATTGGGAAGTGGCATGATGATGACATAGGAGCGGCCTAGGACCCATCCATCTCATGTCTGTCCTGTTGCAGGTGCATCGCCATCTACAGGAGCAGAAGAGTGGACTTGCTACATGACCTAGCATTATTTTCTGGCCCCATTTATCATATCCCTTTTCTCCTCCAAATGTTTCTCCTCTCACCTCTTCTGTGGGACTTAAATTGCTATATCTGCTCAGAGCTCACAAATGCCTTTGAATTATTTCCCTGACTTCCTGATTTTTTTCTTTTCTCAAGTGTTACCTACTAAGGGATGCCTGGAGTAAGCCACCCAGCTACCTAATTCCTCAHLA-DQB1*0201 (Genbank accession number: AY375842.1)HLA-DQB1*0201 allele, exons 1 through 4, and partial cds (SEQ ID NO: 84)TCCCCCTTAATTTGCCCTATTGAAAGAATCCCAAGTATAAGAACAACTGGTTTTTAATCAATATTACAAAGATGTTTACTGTTGAATCGCATTTTTCTTTGGCTTCTTAAAATCCCTTAGGCATTCAATCTTCAGCTCTTCCATAATTGAGAGGAAATTTTCACCTCAAATGTTCATCCAGTGCAATTGAAAGACGTCACAGTGCCAGGCACTGGATTCAGAACCTTCACAAAAAAAAAATCTGCCCAGAGACAGATGAGGTCCTTCAGCTCCAGTGCTGATTGGTTCCTTTCCAAGGGACCATCCAATCCTACCACGCATGGAAACATCCACAGATTTTTATTCTTTCTGCCAGGTACATCAGATCCATCAGGTCCGAGCTGTGTTGACTACCACTTTTCCCTTCGTCTCAATTATGTCTTGGAAAAAGGCTTTGCGGATCCCCGGAGGCCTTCGGGCAGCAACTGTGACCTTGATGCTGTCGATGCTGAGCACCCCAGTGGCTGAGGGCAGAGACTCTCCCGGTAAGTGCAGGGCAGCTGCTCTCCAGAGCCGCTACTCTGGGAACAGGCTCTCCTTGGGCTGGGGTACGGGGATGGTGATCTCCATAATCTCGGACACAATCTTTTATCAACATTTCCTCTGTTTTGGGAAAGAGAGCTATGTTGCATTTCCATTTATCTTTTAATGATGAAGTGAGGACAATCCAATCCCATCCTACAGGCTTAAGCCTGGAAGAGGAGGAGAGAGGAGAGAAAAGAGGAGACAAAGTGTTCATTTACTACCAGTGATAGGACAAAGTGAGCATGGGGTTATTTTTGAAGATATGAATTTCTCCAAAGACACAGCAGGATTTGCCATTTAGGCGTGTCCCAAGACTTGCCTGGACTAAATATTATGATTTCCTGCATTGGGAAATGCAAGGCAGCAATGGTGTCTGTAGTCTCCGTATTTGGGGAAAAGTTGTCTGTATTCCTGACCCAGTGGAGCGTTTGTGGAGGCAAAATCTTGGTACTGAGGGAAGCTGACTGGCTGACCACAGAAAGAGAGCCTTCAGGTTTCACTGATTTATGGGCAAATGGTGACCTGAGTGGGATTCAGATACCCGAGTTGATGATGGACTAAATTTAGTAGAAAGGAGGATGTAAAGAAGGGAAATAACACATACTGTGAAACCACTCATTTCAGACACAGAACAATACTTTACATAAATTCTCTCTCACTCCTTCTAACATCCTGTGTGTAGATATCATGATTTTCTTTTACACAATTATACTTGTGATATGGATATTCTGTTACATAACCTGCCCGGGCTGGTGACTGCCACAGTTTAATGGGAATCTAGTTTATCAAATTCAAAAGCTTGTGCTCTTTCGGTGAATAAATGTTTCTTTCTAGGACTCAGAGATCTAGGACTCCCTTCTTTCTAACACAGACGTGAGTGAACCTCACAGGGCACTTGGGAGGGTAAATCCAGGCATGGGAAGGAAGGTATTTTACCCAGGGACCAAGAGAATAGGCGTATCGGAAGAGGACAGGTTTAATTCCTGGACCTGTCTCGTCATTCCCTTGAACTGTCAGGTTTATGTGGATAACTTTATCTCTGAGGTACCCAGGAGCTCCATGGAAAATGAGATTTCATGCGAGAACGCCCTGATCCCTCTAAGTGCAGAGGTCCATGTAAAATCAGCCCGACTGCCTCTTCACTTGGTTCACAGGCCGAGACAGGGACAGGGCTTTCCTCCCTTTCCTGCCTGTAGGAAGGCGGATTCCCGAAGACCCCCGAGAGGGCGGGCAGGGCTGGGCAGAGCCGCCGGGAGGATCCCAGGTCTGCAGCGCGAGGCACGGGCCGGCGGGAACTTGTGGTCGCGCGGGCTGTTCCACAGCTCCGGGCCGGGTCAGGGTGGCGGCTGCGGGGGCGGACGGGCTGGGCCGCACTGACTGGCCGGTGATTCCTCGCAGAGGATTTCGTGTACCAGTTTAAGGGCATGTGCTACTTCACCAACGGGACAGAGCGCGTGCGTCTTGTGAGCAGAAGCATCTATAACCGAGAAGAGATCGTGCGCTTCGACAGCGACGTGGGGGAGTTCCGGGCGGTGACGCTGCTGGGGCTGCCTGCCGCCGAGTACTGGAACAGCCAGAAGGACATCCTGGAGAGGAAACGGGCGGCGGTGGACAGGGTGTGCAGACACAACTACCAGTTGGAGCTCCGCACGACCTTGCAGCGGCGAGGTGAGCGGCGTCGCCCCTCTGCGAGGCCCACCCTTGGCCCCAAGTCTCTGCGCCAGGAGGGGCGAAGGGTCGTGGCCTCTGGAACCTGAGCCCCGTTTGTTCCACCCCAGAGGACAGGAGGCAGCGGCGAGAGTGGTGGGGGCAGGTGCATCGGAGGTGCGGGGACCTAGGGCAGAGCAGGGGGACAGGCAGAGTTGGCCAGGCTGCCTAGTGTCGCCCCAGCCTACCCGTTCGTCGGCCTTGTCCTCTGCTCTGCATGTTCTTGCCTCGTGCCTTATGCATTTGCCTCCTTTTGCCTTACCTTTGCTAAGCAGCTCTCTCTGCTCAGAATGCCCGCCCTCTTCCCCTGCCCGCCCGCCCGCCCCACTAGCACTGCCCCACCCAGCAAGGCCCACGTGCACAGCTCTTGCAGCAGGAAGCTTCAGGCTTAGCCTGGTGGAGTTAGGGCTGTTCCACAACTGCGCGCAGGACATTCAGCAATTACAGTTGTGAAATAAGATATTTTAACTTTTGGCTTCAAATCATTATTCATCGTAATTCTGTTTTCTTAAATGGCTCTCATTCATGGCAGAGATCTTTGAGGTGAGGGTGTTTTAATCATTGCATGCCTAGTACCTGACACATTGACTGGTATGTGGTGTGAGCTCAATGATCTTCTGTTAAATTAATGAATAAATGTACTCAGCTGCCCATCCACTTAGGCTCAAGAAAAAAAAAGAGGTAAACAGAGCCTTAAAAATGGACTTTATTAATTATTTTCTATAATTTTGCTTAATGCTTTAAAGTAAACTCTTATTGACTTGGATCTTAATAGAGTTTGTGAATACAAAATCTGAGGAAAAAAGTTTTTGCTAAAAATAAAAACAACGCTTGAAAGATATTGTAAGGCAGTTTAAATTTCTTTTCTTTTCTTTTTTTTTTTTTTTGAGACGGATTCTCACTCTGTCGCCCAGGCCGGAGTGCAGTGGCGCGATCTCGGCTCACTGCAAGCTCCGCCTCCCGGGTTCACGCCATTCTCCTGCCTCAGCCTCCTGAGTAGGTGGGATTACAGGCGCGTGCCACCACGCCCGGCTAATTTTTTTGTATTTTTAGTAGAGGCGGGGTTTCACCGTGTTAGCCAGGATGGTCTGGATCTCCTGACCTCATGATCCGCCCGCCTCGGCCTCCCAAAGTGCTGGGATTACAGGTGTGAGCCACAGTGCCCGGCCGGCACTTTTAATTTCTTAGAAAAGCTGAACAAATGGCACAATGCAAAGAGCAAAAGTTTTGGAATAAATAGATTGAAGCCATTAAATTATTGGATAAAAATAGTTTCGGGTTGCTTTTGGCCTAGGTTCTCCCCTCCCCCCATGACTATCCACTTCAGGAATAAACATTCTGAAAGTCAATTTTACCCATTTAGTGAGCATTTATTTCTAGACAGTTGCCTTATCAAATACCATCTATGTTACGTCATTTAATCTCACAGTTACTTGTGCATCAGAGATTAGCATCACCACTTTATATATTGGTACATGATAAACACTTTATTGGTCATGGATGGGGAGATGGTCACTGTAGGCTAATATTGGTACATGATAAACACTTTAAGTAATCAGCCCATAATTGCTCACCAAGACCTTAAGCCTCCCAAAGTACACAACATTCTTTGTGTTCTTCACTACACATCCATAGAGTCTAAGGGACGTAAAGCCTCGTTAAAGCCAGTTTTGACCAGAAGCAGCAATGAGTCTATTCCTGTGTGTTTTCCATGTTAATGGGACAAAATGATACTTTCAAGGCATTGAAAATTCATGATTAATCAATCCCTAGTCTGACCCCAGTGTTATCTATGCAGGTTTGCAAAACCTTTAGTTTACTTAATACTCCCTTGCCTTCTTTTGATTCACATCCTAATGCCAGCAAATACTTATGTTTTTGCTATTTCAGTTCCATTTCCATAAAATTTATTTTATCATCTTTTCTCATAAATTTATGCCCTCTATTTTTACTCCCAATCTGTTTAAGATGAACAAATCTTATAAGGCCACATAGCTGACTGTTATTTCTGTTGGACTCCAGGAAGGAGAACCTAAAGAAAAGTTCAAGTCCAAGCAGAAACCGTGATTTCTTCCAGATGATGGCTCATGAGTGCCATTTAATTGGGGTGCCACCTGGTGACCTCAGCAAATCCCAGCTATATTTATGTGTTCACATTACAGGATCATTAACCCAGACCGACCACTGCACAGATCTCAGAATATTTTCTATGGAGAACATACATAATAATGCCTGATTTCAGAAGAAGAAAGTAATTCTCAATAGCAAGGGGATGGAGTAGGGTAGACAGCTGTAATTAAACTCACTTGTGTGATAAAAAGAAATTAAGGAAAAAAGAAAATGAGAGAACATATTACTAAATAAAGAAAGCATACATTAAATATTTACTATAGTTTCACACTAAGAGAATAAAGGAAATGCAATAAAGTGGCCTGAAAGGTAAAGGATGAGATGTGTAAAGGGGTGTAGTATTTTTACTATGAGCAGCAATCTGAGAAGATAAAGGAATCGAGTTACGGGCAAACATGATGTTTGATCAGTGTTATTTGTTTTCAAGGCCTGCCTAAATTTTTTTCAAATATTACAAACTTTTGAAATAACATTCTTTTTGTTTTTTGCTGTCTGTTACTAGGTTGCACATTTTATAAAGGCAGGGACCATGGTATGTTGTTTGTCTTTGGATTCTCAGTGATTGTTATATTTATATTTGTTGAAGGAACCTTAATCCAAGACTTGGACTCCAAGTATCTTTCCACTCTGGTTCCAAGGAGGGACCTTCCTCACAGCAGGCATGCTGTGTGGTCTCACATCTCACTCCTATATCTTTCCCTGTCTGTTACTGCCCTCAGTGGAGCCCACAGTGACCATCTCCCCATCCAGGACAGAGGCCCTCAACCACCACAACCTGCTGGTCTGCTCGGTGACAGATTTCTATCCAGCCCAGATCAAAGTCCGGTGGTTTCGGAATGACCAGGAGGAGACAGCTGGCGTTGTGTCCACCCCCCTTATTAGGAATGGTGACTGGACCTTCCAGATCCTGGTGATGCTGGAAATGACTCCCCAGCGTGGAGACGTCTACACCTGCCACGTGGAGCACCCCAGCCTCCAGAGCCCCATCACCGTGGAGTGGCGTAAGGGGATATTGAGTTTCTGTTACTGTGGGCCCCACAAGACAAAGGACAGAGCTCCTTCTGACCCATCCCTTCCCATCTCTTATCCCTGATGTCACTGCTGAGCTGGGAATCACAGGAGACTAGAGCACCTCTAGTTCCATGGCGAGTGCATCAGAAGAATCCTGATCTCATCACCTTTCCAGATGCTAGGGAAATTACTCTACATACTGTTGCTCTGGATCCCAGTCCTGATTGCTCTGAGGAACTGATTATTAGGGCTGGTGACTGGGATCTTAGGGTCTAAGTTTATGGATGAGTTCCTGAGGAGTGGAGATCTGCTTCCCCACTCTGTCACCTACTCACTGTATCCAAGGACCTATTGGCTGGCCTTTCCCTCCCTTAGGGGTGGTCTGAATGGAGAACTAGGTTCCTTTGATGCCTTCACCTCCTGCATCTCAGACTGGACTTCAGCTCCTCATCAGGGAAACTATGGGGTATGGGGACAAACACTGACACTCAGGCTCTGCTTCTCAGGGGCTCAATCTGAATCTGCCCAGAGCAAGATGCTGAGTGGCAT HLA-DQB1*0202 (Genbank accession number: AY375844.1)HLA-DQB1*0202 allele, exons 1 through 4, and partial cds (SEQ ID NO: 85)TTGAAAGAATCCCAAGTATAAGAACAACTGGTTTTTAATCAATATTACAAAGATGTTTACTGTTGAATCGCATTTTTCTTTGGCTTCTTAAAATCCCTTAGGCATTCAATCTTCAGCTCTTCCATAATTGAGAGGAAATTTTCACCTCAAATGTTCATCCAGTGCAATTGAAAGACGTCACAGTGCCAGGCACTGGATTCAGAACCTTCACACAAAAAAAATCTGCCCAGAGACAGATGAGGTCCTTCAGCTCCAGTGCTGATTGGTTCCTTTCCAAGGGACCATCCAATCCTACCACGCATGGAAACATCCACAGATTTTTATTCTTTCTGCCAGGTACATCAGATCCATCAGGTCCGAGCTGTGTTGACTACCACTTTTCCCTTCGTCTCAATTATGTCTTGGAAAAAGGCTTTGCGGATCCCCGGAGGCCTTCGGGCAGCAACTGTGACCTTGATGCTGTCGATGCTGAGCACCCCAGTGGCTGAGGGCAGAGACTCTCCCGGTAAGTGCAGGGCAGCTGCTCTCCAGAGCCGCTACTCTGGGAACAGGCTCTCCTTGGGCTGGGGTACGGGGATGGTGATCTCCATAATCTCGGACACAATCTTTTATCAACATTTCCTCTGTTTTGGGAAAGAGAGCTATGTTGCATTTCCATTTATCTTTTAATGATGAAGTGAGGACAATCCAATCCCATCCTACAGGCTTAAGCCTGGAAGAGGAGGAGAGAGGAGAGAAAAGAGGAGACAAAGTGTTCATTTACTACCAGTGATAGGACAAAGTGAGCATGGGGTTATTTTTGAAGATATGAATTTCTCCAAAGACACAGCAGGATTTGCCATTTAGGCGTGTCCCAAGACTTGCCTGGACTAAATATTATGATTTCCTGCATTGGGAAATGCAAGGCAGCAATGGTGTCTGTAGTCTCCGTATTTGGGGAAAAGTTGTCTGTATTCCTGACCCAGTGGAGCGTTTGTGGAGGCAAAATCTTGGTACTGAGGGAAGCTGACTGGCTGACCACAGAAAGAGAGCCTTCAGGTTTCACTGATTTATGGGCAAATGGTGACCTGAGTGGGATTCAGATACCCGAGTTGATGATGGACTAAATTTAGTAGAAAGGAGGATGTAAAGAAGGGAAATAACACATACTGTGAAACCACTCATTTCAGACACAGAACAATACTTTACATAAATTCTCTCTCACTCCTTCTAACATCCTGTGTGTAGATATCATGATTTTCTTTTACACAATTATACTTGTGATATGGATATTCTGTTACATAACCTGCCCGGGCTGGTGACTGCCACAGTTTAATGGGAATCTAGTTTATCAAATTCAAAAGCTTGTGCTCTTTCGGTGAATAAATGTTTCTTTCTAGGACTCAGAGATCTAGGACTCCCTTCTTTCTAACACAGAAGTGAGTGAACCTCACAGGGCACTTGGGAGGGTAAATCCAGGCATGGGAAGGAAGGTATTTTACCCAGGGACCAAGAGAATAGGCGTATCGGAAGAGGACAGGTTTAATTCCTGGACCTGTCTCGTCATTCCCTTGAACTGTCAGGTTTATGTGGATAACTTTATCTCTGAGGTACCCAGGAGCTCCATGGAAAATGAGATTTCATGCGAGAACGCCCTGATCCCTCTAAGTGCAGAGGTCCATGTAAAATCAGCCCGACTGCCTCTTCACTTGGTTCACAGGCCGAGACAGGGACAGGGCTTTCCTCCCTTTCCTGCCTTTAGGAAGGCGGATTCCCGAAGACCCCCGAGAGGGCGGGCAGGGCTGGGCAGAGCCGCCGGGAGGATCCCAGGTCTGCAGCGCGAGGCACGGGCCGGCGGGAACTTGTGGTCGCGCGGGCTGTTCCACAGCTCCGGGCCGGGTCAGGGTGGCGGCTGCGGGGGCGGACGGGCTGGGCCGCACTGACTGGCCGGTGATTCCTCGCAGAGGATTTCGTGTACCAGTTTAAGGGCATGTGCTACTTCACCAACGGGACAGAGCGCGTGCGTCTTGTGAGCAGAAGCATCTATAACCGAGAAGAGATCGTGCGCTTCGACAGCGACGTGGGGGAGTTCCGGGCGGTGACGCTGCTGGGGCTGCCTGCCGCCGAGTACTGGAACAGCCAGAAGGACATCCTGGAGAGGAAACGGGCGGCGGTGGACAGGGTGTGCAGACACAACTACCAGTTGGAGCTCCGCACGACCTTGCAGCGGCGAGGTGAGCGGCGTCGCCCCTCTGCGAGGCCCACCCTTGGCCCCAAGTCTCTGCGCCAGGAGGGGCGAAGGGTCGTTGCCTCTGGAACCTGAGCCCCGTTTGTTCCACCCCAGAGGACAGGAGGCAGCGGCGAGAGTGGTGGGGGCAGGTGCATCGGAGGTGCGGGGACCTAGGGCAGAGCAGGGGGACAGGCAGAGTTGGCCAGGCTGCCTAGTGTCGCCCCAGCCTACCCGTTCGTCGGCCTTGTCCTCTGCTCTGCATGTTCTTGCCTCGTGCCTTATGCATTTGCCTCCTTTTGCCTTACCTTTGCTAAGCAGCTCTCTCTGCTCAGAATGCCCGCCCTCTTCCCCTGCCCGCCCGCCCGCCCCGCTAGCACTGCCCCACCCAGCAAGGCCCACGTGCACAGCTCTTGCAGCAGGAAGCTTCAGGCTTAGCCTGGTGGAGTTAGGGCTGTTCCACAACTGCGCGCAGGACATCCAGCAATTACAGTTGTGAAATAAGATATTTTAACTTTTGGCTTCAAATCATTATTCATCGTAATTCTGTTTTCTTAAATGGCTCTCATTCATGGCAGAGATCTTTGAGGTGAGGGTGTTTTAATCATTGCATGCCTAGTACCTGACACATTGACTGGTATGTGGTGTGAGCTCAATGATCTTCTGTTAAATTAATGAATAAATGTACTCAGCTGCCCATCCACTTAGGCTCAAGAAAAAAAAAGAGGTAAACAGAGCCTTAAAAATGGACTTTATTAATTATTTTCTATAATTTTGCTTAATGCTTTAAAGTAAACTCTTATTGACTTGGATCTTAATAGAGTTTGTGAATACAAAATCTGAGGAAAAAAGTTTTTGCTAAAAATAAAAACAACGCTTGAAAGATATTGTAATGCAGTTTAAATTTCTTTTCTTTTTTTTTTTTTTTTTGAGACGGATTCTCACTCTGTCGCCCAGGCCGGAGTGCAGTGGCGCGATCTCGGCTCACTGCAAGCTCCGCCTCCCGGGTTCACGCCATTCTCCTGCCTCAGCCTCCTGAGTAGGTGGGATTACAGGCGCGTGCCACCACGCCCGGCTAATTTTTTTGTATTTTTAGTAGAGGCGGGGTTTCACCGTGTTAGCCAGGATGGTCTGGATCTCCTGACCTCATGATCCGCCCGCCTCGGCCTCCCAAAGTGCTGGGATTACAGGTGTGAGCCACAGTGCCCGGCCGGCACTTTTAATTTCTTAGAAAAGCTGAACAAATGGCACAATGCAAAGAGCAAAAGTTTTGGAATAAATAGATTGAAGCCATTAAATTATTGGATAAAAATAGTTTCGGGTTGCTTTTGGCCTAGGTTCTCCCCTCCCCCCATGACTATCCACTTCAGGAATAAACATTCTGAAAGTCAATTTTACCCATTTAGTGAGCATTTATTTCTAGACAGTTGCCTTATCAAATACCATCTATGTTACGTCATTTAATCTCACAGTTACTTGTGCATCAGAGATTAGCATCACCACTTTATATATTGGTACATGATAAACACTTTATTGGTCATGGATGGGGAGATGGTCACTGTAGGCTAATATTGGTACATGATAAACACTTTAAGTAATCAGCCCATAATTGCTCACCAAGACCTTAAGCCTCCCAAAGTACACAACATTCTTTGTGTTCTTCACTACACATCCATAGAGTCTAAGGGACGTAAAGCCTCGTTAAAGCCAGTTTTGACCAGAAGCAGCAATGAGTCTATTCCTGTGTGTTTTCCATGTTAATGGGACAAAATGATACTTTCAAGGCATTGAAAATTCATGATTAATCAATCGCTAGTCTGACCCCAGTGTTATCTATGCAGGTTTGCAAAACCTTTAGTTTACTTAATACTCCCTTGCCTTCTTTTGATTCACATCCTAATGCCAGCAAATACTTATGTTTTTGCTATTTCAGTTCCATTTCCATAAAATTTATTTTATCATCTTTTCTCATAAATTTATGCCCTCTATTTTTACTCCCAATCTGTTTAAGATGAACAAATCTTATAAGGCCACATAGCTGACTGTTATTTCTGTTGGACTCCAGGAAGGAGAACCTAAAGAAAAGTTCAAGTCCAAGCAGAAACCGTGATTTCTTCCAGATGATGGCTCATGAGTGCCATTTAATTGGGGTGCCACCTGGTGACCTCAGCAAATCCCAGCTATATTTATGTGTTCACATTACAGGATCATTAACCCAGACCGACCACTGCACAGATCTCAGAATATTTTCTATGGAGAACATACATAATAATGCCTGATTTCAGAAGAAGAAAGTAATTCTCAATAGCAAGGGGATGGAGTAGGGTAGACAGCTGTAATTAAACTCACTTGTGTGATAAAAAGAAATTAAGGAAAAAAGAAAATGAGAGAACATATTACTAAATAAAGAAAGCATACATTAAATATTTACTATAGTTTCACACTAAGAGAATAAAGGAAATGCAATAAAGTGGCCTGAAAGGTAAAGGATGAGATGTGTAAAGGGGTGTAGTATTTTTACTATGAGCAGCAATCTGAGAAGATAAAGGAATCGAGTTACGGGCAAACATGATGTTTGATCAGTGTTATTTGTTTTCAAGGCCTGCCTAAATTTTTTTCAAATATTACAAACTTTTGAAATAACATTCTTTTTGTTTTTTGCTGTCTGTTACTAGGTTGCACATTTTATAAAGGCAGGGACCATGGTATGTTGTTTGTCTTTGGATTCTCAGTGATTGTTATATTTATATTTGTTGAAGGAACCTTAATCCAAGACTTGGACTCCAAGTATCTTTCCACTCTGGTTCCAAGGAGGGACCTTCCTCACAGCAGGCATGCTGTGTGGTCTCACATCTCACTCCTATATCTTTCCCTGTCTGTTACTGCCCTCAGTGGAGCCCACAGTGACCATCTCCCCATCCAGGACAGAGGCCCTCAACCACCACAACCTGCTGGTCTGCTCGGTGACAGATTTCTATCCAGCCCAGATCAAAGTCCGGTGGTTTCGGAATGGCCAGGAGGAGACAGCTGGCGTTGTGTCCACCCCCCTTATTAGGAATGGTGACTGGACCTTCCAGATCCTGGTGATGCTGGAAATGACTCCCCAGCGTGGAGACGTCTACACCTGCCACGTGGAGCACCCCAGCCTCCAGAGCCCCATCACCGTGGAGTGGCGTAAGGGGATATTGAGTTTCTGTTACTGTGGGCCCCACAAGACAAAGGACAGAGCTCCTTCTGACCCATCCCTTCCCATCTCTTATCCCTGATGTCACTGCTGAGCTGGGAATCACAGGAGACTAGAGCACCTCTAGTTCCATGGCGAGTGCATCAGAAGAATCCTGATCTCATCACCTTTCCAGATGCTAGGGAAATTACTCTACATACTGTTGCTCTGGATCCCAGTCCTGATTGCTCTGAGGAACTGATTATTAGGGCTGGTGACTGGGATCTTAGGGTCTAAGTTTATGGATGAGTTCCTGAGGAGTGGAGATCTGCTTCCCCACTCTGTCACCTACTCACTGTATCCAAGTACCTATTGGCTGGCCTTTCCCTCCCTTAGGGGTGGTCTGAATGGAGAACTAGGTTCCTTTGATGCCTTCACCTCCTGCATCTCAGACTGGACTTCAGCTCCTCATCAGGGAAACTATGGGGTATGGGGACAAACACTGACACTCAGGCTCTGCTTCTCAGGGGCTCAATCTGAATCTGCCCAGAGCAAGATGCTGAGTGGCA

HLA Genotype Assays

Other aspects of the disclosure relate to assays for detecting the HLAgenotype of a subject, e.g., a nucleic-acid based assay. The HLAgenotype may be detected, e.g., using one or more single nucleotidepolymorphisms associated with an HLA genotype or by sequencing all orpart of an HLA-DQA and/or HLA-DQB gene. Exemplary SNPs for use in HLAgenotyping include, but are not limited to: rs2187668, rs2395182,rs4713586, rs7775228, rs4639334, and rs7454108. Any one or more of suchexemplary SNPs may be used for HLA genotyping.

Detection of a nucleic acid sequence, e.g., the sequence of an HLA DQAand/or DQB gene, or a portion thereof (e.g., a SNP or a fragment of thegene), may be accomplished using any nucleic-acid based assay known inthe art (see, e.g., Bunce M, et al. Phototyping: comprehensive DNAtyping for HLA-A, B, C, DRB1, DRB3, DRB4, DRB5 & DQB1 by PCR with 144primer mixes utilizing sequence-specific primers (PCR-SSP). TissueAntigens 46, 355-367 (1995); Olerup O, Aldener A, Fogdell A. HLA-DQB1and DQA1 typing by PCR amplification with sequence-specific primers in 2hours. Tissue antigens 41, 119-134 (1993); Mullighan C G, Bunce M, WelshK I. High-resolution HLA-DQB1 typing using the polymerase chain reactionand sequence-specific primers. Tissue-Antigens. 50, 688-92 (1997);Koskinen L, Romanos J, Kaukinen K, Mustalahti K, Korponay-Szabo I, etal. (2009) Cost-effective HLA typing with tagging SNPs predicts celiacdisease risk haplotypes in the Finnish, Hungarian, and Italianpopulations. Immunogenetics 61: 247-256.; and Monsuur A J, de Bakker PI, Zhernakova A, Pinto D, Verduijn W, et al. (2008) Effective detectionof human leukocyte antigen risk alleles in celiac disease using tagsingle nucleotide polymorphisms. PLoS ONE 3: e2270; Koskinen L, RomanosJ, Kaukinen K, Mustalahti K, Korponay-Szabo I, Barisani D, Bardella M T,Ziberna F, Vatta S, Szeles G et al: Cost-effective HLA typing withtagging SNPs predicts Celiac disease risk haplotypes in the Finnish,Hungarian, and Italian populations. Immunogenetics 2009, 61(4):247-256;Monsuur A J, de Bakker P I, Zhernakova A, Pinto D, Verduijn W, RomanosJ, Auricchio R, Lopez A, van Heel D A, Crusius J B et al: Effectivedetection of human leukocyte antigen risk alleles in Celiac diseaseusing tag single nucleotide polymorphisms. PLoS ONE 2008, 3(5):e2270).

Exemplary nucleic acid-based assays include, but are not limited to,PCR, restriction fragment length polymorphism identification (RFLPI),random amplified polymorphic detection (RAPD), amplified fragment lengthpolymorphism detection (AFLPD), allele specific oligonucleotide (ASO)probes, hybridization to microarrays or beads, Sanger sequencing,Single-molecule real-time sequencing (Pacific Bio), Ion semiconductor(Ion Torrent sequencing), Pyrosequencing (454), Single molecule realtime (SMRT) sequencing), Sequencing by synthesis (Illumina), andSequencing by ligation (SOLiD sequencing). The assays may include theuse of one or more nucleic acid probes or primers. The one or moreprobes or primers may be designed, e.g., to specifically bind to nucleicsequences within one or more HLA-DQA or DQB alleles. Methods fordesigning probes and primers are known in the art. The probes or primersmay be attached to a detectable label. Any suitable detectable label iscontemplated. Detectable labels include any composition detectable byspectroscopic, photochemical, biochemical, immunochemical, chemical, orother physical means, e.g., an enzyme, a radioactive label, afluorophore, an electron dense reagent, biotin, digoxigenin, or ahapten. Such detectable labels are well-known in the art and can bedetected through use of, e.g., an enzyme assay, a chromogenic assay, aluminometric assay, a fluorogenic assay, or a radioimmune assay. Thereaction conditions to perform detection of the detectable label dependupon the detection method selected.

Gluten Peptide Treatment

Aspects of the disclosure relate to gluten peptide treatments and usesthereof in any one of the methods described herein. As used herein theterm “gluten peptide” includes any peptide comprising a sequence derivedfrom, or encompassed within, one or more of gluten proteins alpha (α),beta (β), γ (γ) and omega (ω) gliadins, and low and high molecularweight (LMW and HMW) glutenins in wheat, B, C and D hordeins in barley,β, γ and omega secalins in rye, and optionally avenins in oats,including deamidated variants thereof containing one or more glutamineto glutamate substitutions. In some embodiments, the gluten peptide(s)stimulate a CD4+ T cell specific response.

A gluten peptide may include one or more epitopes known to be recognizedby a CD4⁺ T cell in a subject with Celiac disease, e.g., PELP (SEQ IDNO: 12), PELPY (SEQ ID NO: 13), QPELPYP (SEQ ID NO: 64), PQPELPY (SEQ IDNO: 65), FPQPELP (SEQ ID NO: 66), PELPYPQ (SEQ ID NO: 67), FPQPELPYP(SEQ ID NO: 68), PYPQPELPY (SEQ ID NO: 14), PFPQPELPY (SEQ ID NO: 1),PQPELPYPQ (SEQ ID NO: 2), PFPQPEQPF (SEQ ID NO: 3), PQPEQPFPW (SEQ IDNO: 4), PIPEQPQPY (SEQ ID NO: 5), PQPELPYPQ (SEQ ID NO: 2), FRPEQPYPQ(SEQ ID NO: 27), PQQSFPEQQ (SEQ ID NO: 28), IQPEQPAQL (SEQ ID NO: 29),QQPEQPYPQ (SEQ ID NO: 30), SQPEQEFPQ (SEQ ID NO: 31), PQPEQEFPQ (SEQ IDNO: 32), QQPEQPFPQ (SEQ ID NO: 33), PQPEQPFCQ (SEQ ID NO: 34), QQPFPEQPQ(SEQ ID NO: 35), PFPQPEQPF (SEQ ID NO: 3), PQPEQPFPW (SEQ ID NO:4),PFSEQEQPV (SEQ ID NO: 36), FSQQQESPF (SEQ ID NO: 37), PFPQPEQPF (SEQ IDNO:3), PQPEQPFPQ (SEQ ID NO: 38), PIPEQPQPY (SEQ ID NO:5), PFPQPEQPF(SEQ ID NO:3), PQPEQPFPQ (SEQ ID NO:38), PYPEQEEPF (SEQ ID NO: 39),PYPEQEQPF (SEQ ID NO: 40), PFSEQEQPV (SEQ ID NO:36), EGSFQPSQE (SEQ IDNO: 41), EQPQQPFPQ (SEQ ID NO: 42), EQPQQPYPE (SEQ ID NO: 43),QQGYYPTSPQ (SEQ ID NO: 44), EGSFQPSQE (SEQ ID NO:41), PQQSFPEQE (SEQ IDNO: 45), or QGYYPTSPQ (SEQ ID NO: 46) (see, e.g., Sollid L M, Qiao S W,Anderson R P, Gianfrani C, Koning F. Nomenclature and listing of celiacdisease relevant gluten epitopes recognized by CD4⁺ T cells.Immunogenetics. 2012; 64:455-60; PCT Publication Nos.: WO/2001/025793,WO/2003/104273, WO/2005/105129, and WO/2010/060155). Preferably, in someembodiments, the gluten peptides that comprise epitopes such as thoseset forth in SEQ ID NO: 12, 13, etc., also comprise additional aminoacids flanking either or both sides of the epitope. Exemplary glutenpeptides and methods for synthesizing such peptides are known in the art(see, e.g., PCT Publication Nos.: WO/2001/025793, WO/2003/104273,WO/2005/105129, and WO/2010/060155, which are incorporated herein byreference in their entirety). In some embodiments, the gluten peptidecomprises PELP (SEQ ID NO: 12), PELPY (SEQ ID NO: 13), QPELPYP (SEQ IDNO: 64), PQPELPY (SEQ ID NO: 65), FPQPELP (SEQ ID NO: 66), or PELPYPQ(SEQ ID NO: 67) and is at least 8 or 9 amino acids in length.

In some embodiments, one or more glutamate residues of a gluten peptidemay be generated by tissue transglutaminase (tTG) deamidation activityupon one or more glutamine residues of the gluten peptide. Thisdeamidation of glutamine to glutamate can cause the generation of glutenpeptides that can bind to HLA-DQ2 or -DQ8 molecules with high affinity.This reaction may occur in vitro by contacting the gluten peptidecomposition with tTG outside of the subject (e.g., prior to or duringcontact of a gluten peptide composition with a sample comprising T cellsfrom a subject) or in vivo following administration through deamidationvia tTG in the body. Deamidation of a peptide may also be accomplishedby synthesizing a peptide de novo with glutamate residues in place ofone or more glutamine residues, and thus deamidation does notnecessarily require use of tTG. For example, PFPQPQLPY (SEQ ID NO: 15)could become PFPQPELPY (SEQ ID NO: 1) after processing by tTG.Conservative substitution of E with D is also contemplated herein (e.g.,PFPQPELPY (SEQ ID NO: 1) could become PFPQPDLPY (SEQ ID NO: 26).Exemplary peptides including an E to D substitution include peptidescomprising or consisting of PFPQPDLPY (SEQ ID NO: 26), PQPDLPYPQ (SEQ IDNO: 69), PFPQPDQPF (SEQ ID NO: 70), PQPDQPFPW (SEQ ID NO: 71), PIPDQPQPY(SEQ ID NO: 72), LQPFPQPDLPYPQPQ (SEQ ID NO: 73), QPFPQPDQPFPWQP (SEQ IDNO: 74), or PQQPIPDQPQPYPQQ (SEQ ID NO: 75). Such substituted peptidescan be the gluten peptides of any of the methods and compositionsprovided herein. Accordingly, gluten peptides that have not undergonedeamidation are also contemplated herein (e.g., gluten peptidescomprising or consisting of PQLP (SEQ ID NO: 16), PQLPY (SEQ ID NO: 17),QPQLPYP (SEQ ID NO: 76), PQPQLPY (SEQ ID NO: 77), FPQPQLP (SEQ ID NO:78), PQLPYPQ (SEQ ID NO: 79), FPQPQLPYP (SEQ ID NO: 80), PYPQPQLPY (SEQID NO: 18), PFPQPQLPY (SEQ ID NO: 15), PQPQLPYPQ (SEQ ID NO: 19),PFPQPQQPF (SEQ ID NO: 20), PQPQQPFPW (SEQ ID NO: 21), PIPQQPQPY (SEQ IDNO: 22), LQPFPQPQLPYPQPQ (SEQ ID NO: 23), QPFPQPQQPFPWQP (SEQ ID NO:24), or PEQPIPQQPQPYPQQ (SEQ ID NO: 25), PQPQLPYPQ (SEQ ID NO:19),FRPQQPYPQ (SEQ ID NO: 47), PQQSFPQQQ (SEQ ID NO: 48), IQPQQPAQL (SEQ IDNO: 49), QQPQQPYPQ (SEQ ID NO: 50), SQPQQQFPQ (SEQ ID NO: 51), PQPQQQFPQ(SEQ ID NO: 52), QQPQQPFPQ (SEQ ID NO: 53), PQPQQPFCQ (SEQ ID NO: 54),QQPFPQQPQ (SEQ ID NO: 55), PFPQPQQPF (SEQ ID NO:20), PQPQQPFPW (SEQ IDNO: 21), PFSQQQQPV (SEQ ID NO: 56), FSQQQQSPF (SEQ ID NO: 57), PFPQPQQPF(SEQ ID NO:20), PQPQQPFPQ (SEQ ID NO: 58), PIPQQPQPY (SEQ ID NO:22),PFPQPQQPF (SEQ ID NO:20), PQPQQPFPQ (SEQ ID NO:58), PYPEQQEPF (SEQ IDNO: 59), PYPEQQQPF (SEQ ID NO: 60), PFSQQQQPV (SEQ ID NO:56), QGSFQPSQQ(SEQ ID NO: 61), QQPQQPFPQ (SEQ ID NO:53), QQPQQPYPQ (SEQ ID NO:50),QQGYYPTSPQ (SEQ ID NO:53), QGSFQPSQQ (SEQ ID NO:61), PQQSFPQQQ (SEQ IDNO:48), QGYYPTSPQ (SEQ ID NO:56), LQPFPQPELPYPQPQ (SEQ ID NO: 62),QPFPQPQQPFPWQP (SEQ ID NO:24), or PQQPIPQQPQPYPQQ (SEQ ID NO: 63)). Insome embodiments, the gluten peptide comprises PQLP (SEQ ID NO: 16),PQLPY (SEQ ID NO: 17), QPQLPYP (SEQ ID NO: 76), PQPQLPY (SEQ ID NO: 77),FPQPQLP (SEQ ID NO: 78), or PQLPYPQ (SEQ ID NO: 79) and is at least 8 or9 amino acids in length.

A gluten peptide may also be an analog of any of the peptides describedherein. Preferably, in some embodiments the analog is recognized by aCD4⁺ T cell that recognizes one or more of the epitopes listed herein.Exemplary analogs comprise a peptide that has a sequence that is, e.g.,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous to theepitopes specifically recited herein. In some embodiments, the analogscomprise a peptide that is, e.g., 75%, 80%, 85%, 90%, 95%, 96%, 97%,98%, or 99% homologous to the peptides specifically recited herein.Analogs may also be a variant of any of the peptides provided, suchvariants can include conservative amino acid substitution variants,e.g., E to D substitution.

In some embodiments, analogs may include one or more amino acidsubstitutions as shown in Table 1 (see, e.g., Anderson et al.Antagonists and non-toxic variants of the dominant wheat gliadin T cellepitope in coeliac disease. Gut. 2006 April; 55(4): 485-491; and PCTPublication WO2003104273, the contents of which are incorporated hereinby reference). The gluten peptides provided herein include analogs ofSEQ ID NO:68 comprising one or more of the listed amino acidsubstitutions. In some embodiments, the analog is an analog of SEQ IDNO: 68 comprising one of the amino acid substitutions provided in Table1 below. Preferably, analogs generate a T cell response as describedherein.

TABLE 1 Exemplary substitutions in the epitope FPQPELPYP (SEQ ID NO: 68)Amino acid in epitope F P Q P E L P Y P Exemplary A, G, H, I,A, F, I, M, A, F, G, — D M S I, S, S, T, Substitutions L, M P, S,S, T, V, H, I, L, V, W Y T, W, Y W, Y M, S, T, V

The length of the peptide may vary. In some embodiments, peptides are,e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more amino acids inlength. In some embodiments, peptides are, e.g., 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 60, 70, 80, 90, or 100 or fewer amino acids in length. Insome embodiments, peptides are, e.g., 4-1000, 4-500, 4-100, 4-50, 4-40,4-30, or 4-20 amino acids in length. In some embodiments, peptides are4-20, 5-20, 6-20, 7-20, 8-20, 9-20, 10-20, 11-20, 12-20, 13-20, 14-20,or 15-20 amino acids in length. In some embodiments, peptides are e.g.,5-30, 10-30, 15-30 or 20-30 amino acids in length. In some embodiments,peptides are 4-50, 5-50, 6-50, 7-50, 8-50, 9-50, 10-50, 11-50, 12-50,13-50, 14-50, or 15-50 amino acids in length. In some embodiments,peptides are 8-50 amino acids in length.

In some embodiments, the gluten peptide treatment is a compositioncomprising at least one or one or more gluten peptide(s). In someembodiments, any one of the methods described herein comprisesadministering the composition to a subject (e.g., a subject having orsuspected of having Celiac disease).

In some embodiments, the gluten peptide treatment comprises acomposition comprising at least one peptide comprising at least oneamino acid sequence selected from PFPQPELPY (SEQ ID NO: 1), PQPELPYPQ(SEQ ID NO: 2), PFPQPEQPF (SEQ ID NO: 3), PQPEQPFPW (SEQ ID NO: 4),PIPEQPQPY (SEQ ID NO: 5) and EQPIPEQPQ (SEQ ID NO: 6). In someembodiments, the gluten peptide treatment comprises a compositioncomprising at least one of: (i) a first peptide comprising the aminoacid sequence PFPQPELPY (SEQ ID NO: 1) and PQPELPYPQ (SEQ ID NO: 2),(ii) a second peptide comprising the amino acid sequence PFPQPEQPF (SEQID NO: 3) and PQPEQPFPW (SEQ ID NO: 4), and (iii) a third peptidecomprising the amino acid sequence PIPEQPQPY (SEQ ID NO: 5) andEQPIPEQPQ (SEQ ID NO: 6). “First”, “second”, and “third” are not meantto imply an order of use or importance, unless specifically statedotherwise. In some embodiments, the composition comprises the first andsecond peptide, the first and third peptide, or the second and thirdpeptide. In some embodiments, the composition comprises the first andsecond peptide. In some embodiments, the composition comprises thefirst, second, and third peptide. In some embodiments, the first peptidecomprises the amino acid sequence LQPFPQPELPYPQPQ (SEQ ID NO: 62); thesecond peptide comprises the amino acid sequence QPFPQPEQPFPWQP (SEQ IDNO: 7); and/or the third peptide comprises the amino acid sequencePEQPIPEQPQPYPQQ (SEQ ID NO: 8).

In some embodiments, it may be desirable to utilize the non-deamidatedforms of such peptides, e.g., if the peptides are contained within acomposition for administration to a subject where tissuetransglutaminase will act in situ (see, e.g., oyvind Molberg, StephenMcAdam, Knut E. A. Lundin, Christel Kristiansen, Helene Arentz-Hansen,Kjell Kett and Ludvig M. Sollid. T cells from celiac disease lesionsrecognize gliadin epitopes deamidated in situ by endogenous tissuetransglutaminase. Eur. J. Immunol. 2001. 31: 1317-1323). Accordingly, insome embodiments, the composition comprises at least one of: (i) a firstpeptide comprising the amino acid sequence PFPQPQLPY (SEQ ID NO: 15) andPQPQLPYPQ (SEQ ID NO: 19), (ii) a second peptide comprising the aminoacid sequence PFPQPQQPF (SEQ ID NO: 20) and PQPQQPFPW (SEQ ID NO: 21),and (iii) a third peptide comprising the amino acid sequence PIPQQPQPY(SEQ ID NO: 22). In some embodiments, the first peptide comprises theamino acid sequence LQPFPQPQLPYPQPQ (SEQ ID NO:23); the second peptidecomprises the amino acid sequence QPFPQPQQPFPWQP (SEQ ID NO:24); and/orthe third peptide comprises the amino acid sequence PQQPIPQQPQPYPQQ (SEQID NO: 63). In some embodiments, the peptides are 8-30 amino acids inlength.

Modifications to a gluten peptide are also contemplated herein. Thismodification may occur during or after translation or synthesis (forexample, by farnesylation, prenylation, myristoylation, glycosylation,palmitoylation, acetylation, phosphorylation (such as phosphotyrosine,phosphoserine or phosphothreonine), amidation, pyrolation,derivatisation by known protecting/blocking groups, proteolyticcleavage, linkage to an antibody molecule or other cellular ligand, andthe like). Any of the numerous chemical modification methods knownwithin the art may be utilized including, but not limited to, specificchemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8protease, NaBH4, acetylation, formylation, oxidation, reduction,metabolic synthesis in the presence of tunicamycin, etc.

The phrases “protecting group” and “blocking group” as used herein,refers to modifications to the peptide, which protect it fromundesirable chemical reactions, particularly chemical reactions in vivo.Examples of such protecting groups include esters of carboxylic acidsand boronic acids, ethers of alcohols and acetals, and ketals ofaldehydes and ketones. Examples of suitable groups include acylprotecting groups such as, for example, furoyl, formyl, adipyl, azelayl,suberyl, dansyl, acetyl, theyl, benzoyl, trifluoroacetyl, succinyl andmethoxysuccinyl; aromatic urethane protecting groups such as, forexample, benzyloxycarbonyl (Cbz); aliphatic urethane protecting groupssuch as, for example, t-butoxycarbonyl (Boc) or9-fluorenylmethoxy-carbonyl (FMOC); pyroglutamate and amidation. Manyother modifications providing increased potency, prolonged activity,ease of purification, and/or increased half-life will be known to theperson skilled in the art.

The peptides may comprise one or more modifications, which may benatural post-translation modifications or artificial modifications. Themodification may provide a chemical moiety (typically by substitution ofa hydrogen, for example, of a C—H bond), such as an amino, acetyl, acyl,amide, carboxy, hydroxy or halogen (for example, fluorine) group, or acarbohydrate group. Typically, the modification may be present on the N-and/or C-terminus. Furthermore, one or more of the peptides may bePEGylated, where the PEG (polyethyleneoxy group) provides for enhancedlifetime in the blood stream. One or more of the peptides may also becombined as a fusion or chimeric protein with other proteins, or withspecific binding agents that allow targeting to specific moieties on atarget cell.

A gluten peptide may also be chemically modified at the level of aminoacid side chains, of amino acid chirality, and/or of the peptidebackbone.

Particular changes can be made to a gluten peptide to improve resistanceto degradation or optimize solubility properties or otherwise improvebioavailability compared to the parent gluten peptide, thereby providinggluten peptides having similar or improved therapeutic, diagnosticand/or pharmacokinetic properties. A preferred such modification, insome embodiments, includes the use of an N-terminal acetyl group orpyroglutamate and/or a C-terminal amide. Such modifications have beenshown in the art to significantly increase the half-life andbioavailability of peptides compared to the peptides having a free N-and C-terminus (see, e.g., PCT Publication No.: WO/2010/060155). In someembodiments, the first, second and/or third peptides comprise anN-terminal acetyl group or pyroglutamate group and/or a C-terminal amidegroup. In some embodiments, the first peptide comprises the amino acidsequence ELQPFPQPELPYPQPQ (SEQ ID NO: 9), wherein the N-terminal E is apyroglutamate; the second peptide comprises the amino acid sequenceEQPFPQPEQPFPWQP (SEQ ID NO: 10), wherein the N-terminal E is apyroglutamate; and/or the third peptide comprises the amino acidsequence EPEQPIPEQPQPYPQQ (SEQ ID NO: 11), wherein the N-terminal E is apyroglutamate. In some embodiments, the first peptide comprises theamino acid sequence ELQPFPQPELPYPQPQ (SEQ ID NO: 9), wherein theN-terminal E is a pyroglutamate, and wherein the peptide contains aC-terminal amide group; the second peptide comprises the amino acidsequence EQPFPQPEQPFPWQP (SEQ ID NO: 10), wherein the N-terminal E is apyroglutamate, and wherein the peptide contains a C-terminal amidegroup; and/or the third peptide comprises the amino acid sequenceEPEQPIPEQPQPYPQQ (SEQ ID NO: 11), wherein the N-terminal E is apyroglutamate, and wherein the peptide contains a C-terminal amidegroup. In some embodiments, the first peptide consists of the amino acidsequence ELQPFPQPELPYPQPQ (SEQ ID NO: 9), wherein the N-terminal E is apyroglutamate, and wherein the peptide contains a C-terminal amidegroup; the second peptide consists of the amino acid sequenceEQPFPQPEQPFPWQP (SEQ ID NO: 10), wherein the N-terminal E is apyroglutamate, and wherein the peptide contains a C-terminal amidegroup; and/or the third peptide consists of the amino acid sequenceEPEQPIPEQPQPYPQQ (SEQ ID NO: 11), wherein the N-terminal E is apyroglutamate, and wherein the peptide contains a C-terminal amidegroup.

In a particular embodiment, a composition comprising a first peptidecomprising the amino acid sequence ELQPFPQPELPYPQPQ (SEQ ID NO:9),wherein the N-terminal glutamate is a pyroglutamate and the C-terminalglutamine is amidated (i.e., the free C-terminal COO is amidated); asecond peptide comprising the amino acid sequence EQPFPQPEQPFPWQP (SEQID NO: 10), wherein the N-terminal glutamate is a pyroglutamate and theC-terminal proline is amidated (i.e., the free C-terminal COO isamidated); and a third peptide comprising the amino acid sequenceEPEQPIPEQPQPYPQQ (SEQ ID NO: 11), wherein the N-terminal glutamate is apyroglutamate and the C-terminal glutamine is amidated (i.e., the freeC-terminal COO is amidated) is contemplated. In some embodiments, thefirst, second and/or third peptides consist essentially of or consist ofthe amino acid sequence of SEQ ID NO: 9, 10, or 11, respectively.Compositions are further described herein.

In another embodiment, a composition comprising first peptide comprisingthe amino acid sequence PFPQPELPY (SEQ ID NO: 1) and the amino acidsequence PQPELPYPQ (SEQ ID NO:2), optionally wherein the N-terminuscomprises a pyroglutamate (e.g., any N-terminal glutamate is apyroglutamate) and the C-terminus is amidated (e.g., any C-terminalglutamine is amidated); a second peptide comprising the amino acidsequence PFPQPEQPF (SEQ ID NO:3) and the amino acid sequence PQPEQPFPW(SEQ ID NO:4), optionally wherein the N-terminus comprises apyroglutamate (e.g., any N-terminal glutamate is a pyroglutamate) andthe C-terminus is amidated (e.g., any C-terminal proline is amidated);and a third peptide comprising the amino acid sequence EQPIPEQPQ (SEQ IDNO:6) and the amino acid sequence PIPEQPQPY (SEQ ID NO:5), optionallywherein the N-terminus comprises a pyroglutamate (e.g., any N-terminalglutamate is a pyroglutamate) and the C-terminus is amidated (e.g., anyC-terminal glutamine is amidated) is contemplated.

Certain peptides described herein may exist in particular geometric orstereoisomeric forms. The present disclosure contemplates all suchforms, including cis-(Z) and trans-(E) isomers, R- and S-enantiomers,diastereomers, (D)-isomers, (L)-isomers, the racemic mixtures thereof,and other mixtures thereof, as, falling within the scope of thedisclosure. Additional asymmetric carbon atoms may be present in asubstituent, such as an alkyl group. All such isomers, as well asmixtures thereof, are intended to be included in this disclosure.

In another example, to prevent cleavage by peptidases, any one or moreof the peptides may include a non-cleavable peptide bond in place of aparticularly sensitive peptide bond to provide a more stable peptide.Such non-cleavable peptide bonds may include beta amino acids.

In certain embodiments, any one or more of the peptides may include afunctional group, for example, in place of the scissile peptide bond,which facilitates inhibition of a serine-, cysteine- or aspartate-typeprotease, as appropriate. For example, the disclosure includes apeptidyl diketone or a peptidyl keto ester, a peptide haloalkylketone, apeptide sulfonyl fluoride, a peptidyl boronate, a peptide epoxide, apeptidyl diazomethane, a peptidyl phosphonate, isocoumarins,benzoxazin-4-ones, carbamates, isocyantes, isatoic anhydrides or thelike. Such functional groups have been provided in other peptidemolecules, and general routes for their synthesis are known.

The peptides may be in a salt form, preferably, a pharmaceuticallyacceptable salt form. “A pharmaceutically acceptable salt form” includesthe conventional non-toxic salts or quaternary ammonium salts of apeptide, for example, from non-toxic organic or inorganic acids.Conventional non-toxic salts include, for example, those derived frominorganic acids such as hydrochloride, hydrobromic, sulphuric, sulfonic,phosphoric, nitric, and the like; and the salts prepared from organicacids such as acetic, propionic, succinic, glycolic, stearic, lactic,malic, tartaric, citric, ascorbic, palmitic, maleic, hydroxymaleic,phenylacetic, glutamic, benzoic, salicyclic, sulfanilic,2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethanedisulfonic, oxalic, isothionic, and the like.

Peptide Production

The peptides described herein (e.g., gluten peptides) can be prepared inany suitable manner. For example, the peptides can be recombinantlyand/or synthetically produced.

The peptides may be synthesised by standard chemistry techniques,including synthesis by an automated procedure using a commerciallyavailable peptide synthesiser. In general, peptides may be prepared bysolid-phase peptide synthesis methodologies which may involve couplingeach protected amino acid residue to a resin support, preferably a4-methylbenzhydrylamine resin, by activation withdicyclohexylcarbodiimide to yield a peptide with a C-terminal amide.Alternatively, a chloromethyl resin (Merrifield resin) may be used toyield a peptide with a free carboxylic acid at the C-terminal. After thelast residue has been attached, the protected peptide-resin is treatedwith hydrogen fluoride to cleave the peptide from the resin, as well asdeprotect the side chain functional groups. Crude product can be furtherpurified by gel filtration, high pressure liquid chromatography (HPLC),partition chromatography, or ion-exchange chromatography.

If desired, and as outlined above, various groups may be introduced intothe peptide of the composition during synthesis or during expression,which allow for linking to other molecules or to a surface. For example,cysteines can be used to make thioethers, histidines for linking to ametal ion complex, carboxyl groups for forming amides or esters, aminogroups for forming amides, and the like.

The peptides may also be produced using cell-free translation systems.Standard translation systems, such as reticulocyte lysates and wheatgerm extracts, use RNA as a template; whereas “coupled” and “linked”systems start with DNA templates, which are transcribed into RNA thentranslated.

Alternatively, the peptides may be produced by transfecting host cellswith expression vectors that comprise a polynucleotide(s) that encodesone or more peptides.

For recombinant production, a recombinant construct comprising asequence which encodes one or more of the peptides is introduced intohost cells by conventional methods such as calcium phosphatetransfection, DEAE-dextran mediated transfection, microinjection,cationic lipid-mediated transfection, electroporation, transduction,scrape lading, ballistic introduction or infection.

One or more of the peptides may be expressed in suitable host cells,such as, for example, mammalian cells (for example, COS, CHO, BHK, 293HEK, VERO, HeLa, HepG2, MDCK, W138, or NIH 3T3 cells), yeast (forexample, Saccharomyces or Pichia), bacteria (for example, E. coli, P.pastoris, or B. subtilis), insect cells (for example, baculovirus in Sf9cells) or other cells under the control of appropriate promoters usingconventional techniques. Following transformation of the suitable hoststrain and growth of the host strain to an appropriate cell density, thecells can be harvested by centrifugation, disrupted by physical orchemical means, and the resulting crude extract retained for furtherpurification of the peptide or variant thereof.

Suitable expression vectors include, for example, chromosomal,non-chromosomal and synthetic polynucleotides, for example, derivativesof SV40, bacterial plasmids, phage DNAs, yeast plasmids, vectors derivedfrom combinations of plasmids and phage DNAs, viral DNA such as vacciniaviruses, adenovirus, adeno-associated virus, lentivirus, canary poxvirus, fowl pox virus, pseudorabies, baculovirus, herpes virus andretrovirus. The polynucleotide may be introduced into the expressionvector by conventional procedures known in the art.

The polynucleotide which encodes one or more peptides may be operativelylinked to an expression control sequence, i.e., a promoter, whichdirects mRNA synthesis. Representative examples of such promotersinclude the LTR or SV40 promoter, the E. coli lac or trp, the phagelambda PL promoter and other promoters known to control expression ofgenes in prokaryotic or eukaryotic cells or in viruses. The expressionvector may also contain a ribosome binding site for translationinitiation and a transcription terminator. The expression vectors mayalso include an origin of replication and a selectable marker, such asthe ampicillin resistance gene of E. coli to permit selection oftransformed cells, i.e., cells that are expressing the heterologouspolynucleotide. The nucleic acid molecule encoding one or more of thepeptides may be incorporated into the vector in frame with translationinitiation and termination sequences.

One or more of the peptides can be recovered and purified fromrecombinant cell cultures (i.e., from the cells or culture medium) bywell-known methods including ammonium sulphate or ethanol precipitation,acid extraction, anion or cation exchange chromatography,phosphocellulose chromatography, hydrophobic interaction chromatography,affinity chromatography, hydroxyapatite chromatography, lectinchromatography, and HPLC. Well known techniques for refolding proteinsmay be employed to regenerate active conformation when the peptide isdenatured during isolation and or purification.

To produce a glycosylated peptide, it is preferred in some embodimentsthat recombinant techniques be used. To produce a glycosylated peptide,it is preferred in some embodiments that mammalian cells such as, COS-7and Hep-G2 cells be employed in the recombinant techniques.

The peptides can also be prepared by cleavage of longer peptides,especially from food extracts.

Pharmaceutically acceptable salts of the peptides can be synthesisedfrom the peptides which contain a basic or acid moiety by conventionalchemical methods. Generally, the salts are prepared by reacting the freebase or acid with stoichiometric amounts or with an excess of thedesired salt-forming inorganic or organic acid or base in a suitablesolvent. In some embodiments, the pharmaceutically acceptable salt is atrifluoroacetate (TFA) salt or an acetate salt.

Dosage and Administration Compositions

The disclosure also provides compositions comprising gluten peptides,e.g., for treatment, for diagnostic methods, for therapeutic efficacymethods, among others. In some embodiments, the composition comprisinggluten peptides is a gluten peptide treatment. In some embodiments, thecomposition comprising gluten peptides is a first composition. In someembodiments, the composition comprising gluten peptides is a secondcomposition.

In some embodiments, the composition comprises a first peptidecomprising the amino acid sequence ELQPFPQPELPYPQPQ (SEQ ID NO:9),wherein the N-terminal glutamate is a pyroglutamate and the carboxylgroup of the C-terminal glutamine is amidated; a second peptidecomprising the amino acid sequence EQPFPQPEQPFPWQP (SEQ ID NO: 10),wherein the N-terminal glutamate is a pyroglutamate and the carboxylgroup of the C-terminal proline is amidated; and a third peptidecomprising the amino acid sequence EPEQPIPEQPQPYPQQ (SEQ ID NO: 11),wherein the N-terminal glutamate is a pyroglutamate and the carboxylgroup of the C-terminal glutamine is amidated. In some embodiments, thecomposition is a vaccine composition.

The disclosure additionally provides a composition comprising a firstpeptide comprising the amino acid sequence PFPQPELPY (SEQ ID NO: 1) andthe amino acid sequence PQPELPYPQ (SEQ ID NO: 2), optionally wherein theN-terminus comprises a pyroglutamate (e.g., any N-terminal glutamate isa pyroglutamate) and the C-terminus is amidated (e.g., any C-terminalglutamine is amidated); a second peptide comprising the amino acidsequence PFPQPEQPF (SEQ ID NO: 3) and the amino acid sequence PQPEQPFPW(SEQ ID NO:4), optionally wherein the N-terminus comprises apyroglutamate (e.g., any N-terminal glutamate is a pyroglutamate) andthe C-terminus is amidated (e.g., any C-terminal proline is amidated);and a third peptide comprising the amino acid sequence EQPIPEQPQ (SEQ IDNO:6) and the amino acid sequence PIPEQPQPY (SEQ ID NO:5), optionallywherein the N-terminus comprises a pyroglutamate (e.g, any N-terminalglutamate is a pyroglutamate) and the C-terminus is amidated (e.g., anyC-terminal glutamine is amidated). In some embodiments, the compositionis a vaccine composition.

As used herein, the term “vaccine” refers to a composition comprisingpeptide(s) that can be administered to a subject having Celiac diseaseto modulate the subject's response to gluten. The vaccine may reduce theimmunological reactivity of a subject towards gluten. Preferably, thevaccine induces tolerance to gluten.

Without being bound by any theory, administration of the vaccinecomposition to a subject may induce tolerance by clonal deletion ofgluten-specific effector T cell populations, for example,gluten-specific CD4⁺ T cells, or by inactivation (anergy) of said Tcells such that they become less responsive, preferably, unresponsive tosubsequent exposure to gluten (or peptides thereof). Deletion orinactivation of said T cells can be measured, for example, by contactingex vivo a sample comprising said T cells with gluten or a peptidethereof and measuring the response of said T cells to the gluten orpeptide thereof. An exemplary T cell response measurement is measurementof the level of interferon-gamma (IFN-γ, see, e.g., NCBI Gene ID 3458and Protein ID NP_000610.2) in the sample after contact with the glutenor peptide thereof. A decreased level of IFN-γ may indicate deletion orinactivation of said T cells. The level of IFN-γ can be measured usingany method known to those of skill in the art, e.g., using immuno-baseddetection methods such as Western blot or enzyme-linked immunosorbentassay (ELISA).

Alternatively, or in addition, administration of the vaccine compositionmay modify the cytokine secretion profile of the subject (for example,result in decreased IL-4, IL-2, TNF-α and/or IFN-γ, and/or increasedIL-10). The vaccine composition may induce suppressor T cellsubpopulations, for example Treg cells, to produce IL-10 and/or TGF-βand thereby suppress gluten-specific effector T cells. The cytokinesecretion profile of the subject can be measured using any method knownto those of skill in the art, e.g., using immuno-based detection methodssuch as Western blot or enzyme-linked immunosorbent assay (ELISA).

The vaccine composition of the disclosure can be used for prophylactictreatment of a subject capable of developing Celiac disease and/or usedin ongoing treatment of a subject who has Celiac disease. In someembodiments, the composition is for use in treating Celiac disease in asubject.

Dosage

The actual amount administered (e.g., dose or dosage) and the rate andtime-course of administration of the gluten peptide composition maydepend upon the HLA genotype of the subject. In some embodiments of anyone of the methods described herein, the method comprises adjusting orselecting a dose of a gluten peptide composition, e.g., gluten peptidetreatment for a subject based on the HLA genotype of the subject. Insome embodiments of any one of the methods described herein, the methodcomprises decreasing a dose of the gluten peptide peptide composition,e.g., gluten peptide treatment if the subject has a homozygous HLA-DQ2.5genotype or maintaining or increasing the dose of the gluten peptidetreatment if the subject has a non-homozygous HLA-DQ2.5 genotype.

In some embodiments of any one of the methods described herein, themethod comprises measuring a level of at least one circulating cytokineor chemokine in a subject that has or is suspected of having Celiacdisease, wherein the subject has been administered a first compositioncomprising at least one gluten peptide in an amount selected based on ahuman leukocyte antigen (HLA) genotype of the subject, and assessing thelikelihood the subject has Celiac disease.

In some embodiments of any one of the methods described herein, themethod comprises assessing tolerance to a gluten peptide in a subjecthaving Celiac disease, the method comprising: measuring a level of atleast one circulating cytokine or chemokine in a subject having Celiacdisease, wherein the subject has been administered a first compositioncomprising at least one gluten peptide in an amount selected based on ahuman leukocyte antigen (HLA) genotype of the subject, and assessing thetolerance of the subject to the at least one gluten peptide based on themeasuring.

HLA genotypes are further described herein. The dose may be decreased,e.g., by decreasing the amount of gluten peptide treatment administeredto the subject or by decreasing the rate of administration of the glutenpeptide treatment to the subject (e.g., by separating eachadministration by a longer period of time).

In some embodiments, the dose is adjusted or selected for a subject suchthat the amount is sufficient to provide the desired therapeutic orphysiological effect when administered under appropriate or sufficientconditions without causing severe adverse effects. In some embodiments,when the gluten peptide treatment is a composition comprising a firstpeptide comprising the amino acid sequence ELQPFPQPELPYPQPQ (SEQ IDNO:9), wherein the N-terminal glutamate is a pyroglutamate and thecarboxyl group of the C-terminal glutamine is amidated; a second peptidecomprising the amino acid sequence EQPFPQPEQPFPWQP (SEQ ID NO: 10),wherein the N-terminal glutamate is a pyroglutamate and the carboxylgroup of the C-terminal proline is amidated; and a third peptidecomprising the amino acid sequence EPEQPIPEQPQPYPQQ (SEQ ID NO: 11),wherein the N-terminal glutamate is a pyroglutamate and the carboxylgroup of the C-terminal glutamine is amidated, the dose to be adjustedis 150 micrograms of the peptides provided herein (i.e., 50 microgramsof the first peptide and an equimolar amount of each of the second andthird peptides). In some embodiments, the dose to be adjusted is 26.5nmol of each of the first, second, and third peptides. Methods forproducing equimolar peptide compositions are known in the art andprovided herein (see, e.g., Example 1 and Muller et al. Successfulimmunotherapy with T-cell epitope peptides of bee venom phospholipase A2induces specific T-cell anergy in patient allergic to bee venom. J.Allergy Clin. Immunol. Vol. 101, Number 6, Part 1: 747-754 (1998)). Insome embodiments, the dose to be adjusted is 300 micrograms of thepeptides provided herein (i.e., 100 micrograms of the first peptide andan equimolar amount of each of the second and third peptides). In someembodiments, the dose to be adjusted is administered in sterile sodiumchloride 0.9% USP as a bolus intradermal injection.

In some embodiments of any one of the methods provided, the dose is oris decreased to less than 300 micrograms of the peptides if the subjecthas a homozygous HLA-DQ2.5 genotype. In some embodiments of any one ofthe methods provided, the dose is or is decreased to less than 150micrograms if the subject has a homozygous HLA-DQ2.5 genotype. In someembodiments of any one of the methods provided, the dose is or isincreased to up to 300 micrograms if the subject has a heterozygousHLA-DQ2.5 genotype.

In some embodiments of any one of the methods provided, the dose isselected to be up to 300 micrograms if the subject has a heterozygousHLA-DQ2.5 genotype. In some embodiments of any one of the methodsprovided herein, the amount selected based on HLA-DQ2.5 genotype is anyone of the foregoing. In some embodiments, the selected dose for asubject having a homozygous DQ2.5 genotype is less than the dose thatwould be selected for a subject having a heterozygous DQ2.5 genotype. Insome embodiments, the selected dose for a subject having a heterozygousDQ2.5 genotype is more than the dose that would be selected for asubject having a homozygous DQ2.5 genotype.

In some embodiments, the dose that is adjusted or selected for a subjectis believed to modify a T cell response, e.g., by inducing immunetolerance, to wheat, barley and rye in the subject, and preferablywheat, barley, rye and oats. Thus, a subject treated according to thedisclosure preferably is able to eat at least wheat, rye, barley andoptionally oats without a significant T cell response which wouldnormally lead to clinical manifestations of active Celiac disease.

In some embodiments, it is advantageous to formulate the active in adosage unit form for ease of administration and uniformity of dosage.“Dosage unit form” as used herein refers to physically discrete unitssuited as unitary dosages for the subject to be treated; each unitcontaining a predetermined quantity of active agent calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical carrier. The specification for the dosage unit forms aredictated by and directly dependent on the unique characteristics of theactive agent and the particular therapeutic effect to be achieved, andthe limitations inherent in the art of compounding such an active agentfor the treatment of subjects. Examples of dosage units include sealedampoules and vials and may be stored in a freeze-dried conditionrequiring only the addition of the sterile liquid carrier immediatelyprior to use.

The composition may also be included in a container, pack, or dispensertogether with instructions for administration.

The actual amount administered (or dose or dosage) and the rate andtime-course of administration are as provided herein.

The administration may occur at least once, e.g., once or twice a week.In some embodiments, a composition described herein is administered onceor twice a week. In some embodiments, a composition described herein isadministered for 3, 4 or 8 weeks. In some embodiments, a compositiondescribed herein is administered once a week for 8 weeks. In someembodiments, a composition described herein is administered once a weekfor 3 weeks. In some embodiments, a composition described herein isadministered twice a week for 4 weeks. In some embodiments, acomposition described herein is administered twice a week for 8 weeks.In some embodiments, the administration occurs 3, 8 or 16 times.

Kits

Another aspect of the disclosure relates to kits. In some embodiments,the kit comprises a composition comprising the peptides as describedherein. The peptides can be contained within the same container orseparate containers. In some embodiments, the kit can further comprise aplacebo. In some embodiments, the peptide or ptides may be containedwithin the container(s) (e.g., dried onto the wall of the container(s)).In some embodiments, the peptides are contained within a solutionseparate from the container, such that the peptides may be added to thecontainer at a subsequent time. In some embodiments, the peptides are inlyophilized form in a separate container, such that the peptides may bereconstituted and added to the container at a subsequent time.

In some embodiments, the kit further comprises instructions forreconstitution, mixing, administration, etc. In some embodiments, theinstructions include the methods described herein. Instructions can bein any suitable form, e.g., as a printed insert or a label.

Pharmaceutically Acceptable Carriers

The composition may include a pharmaceutically acceptable carrier. Theterm “pharmaceutically acceptable carrier” refers to molecular entitiesand compositions that do not produce an allergic, toxic or otherwiseadverse reaction when administered to a subject, particularly a mammal,and more particularly a human. The pharmaceutically acceptable carriermay be solid or liquid. Useful examples of pharmaceutically acceptablecarriers include, but are not limited to, diluents, excipients,solvents, surfactants, suspending agents, buffering agents, lubricatingagents, adjuvants, vehicles, emulsifiers, absorbants, dispersion media,coatings, stabilizers, protective colloids, adhesives, thickeners,thixotropic agents, penetration agents, sequestering agents, isotonicand absorption delaying agents that do not affect the activity of theactive agents of the disclosure. In some embodiments, thepharmaceutically acceptable carrier is a sodium chloride solution (e.g.,sodium chloride 0.9% USP).

The carrier can be any of those conventionally used and is limited onlyby chemico-physical considerations, such as solubility and lack ofreactivity with the active agent, and by the route of administration.Suitable carriers for this disclosure include those conventionally used,for example, water, saline, aqueous dextrose, lactose, Ringer'ssolution, a buffered solution, hyaluronan, glycols, starch, cellulose,glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silicagel, magnesium stearate, sodium stearate, glycerol monostearate, sodiumchloride, glycerol, propylene glycol, water, ethanol, and the like.Liposomes may also be used as carriers.

Techniques for preparing pharmaceutical compositions are generally knownin the art as exemplified by Remington's Pharmaceutical Sciences, 16thEd. Mack Publishing Company, 1980.

Administration preferably is intradermal administration. Thus, thecomposition of the disclosure may be in a form suitable for intradermalinjection. In some embodiments, the composition of the disclosure is inthe form of a bolus for intradermal injection.

Injectables

The pharmaceutical composition(s) may be in the form of a sterileinjectable aqueous or oleagenous suspension. In some embodiments, thecomposition is formulated as a sterile, injectable solution. Thissuspension or solution may be formulated according to known methodsusing those suitable dispersing or wetting agents and suspending agentswhich have been mentioned above. The sterile injectable preparation maybe a suspension in a non-toxic parenterally-acceptable diluent orsolvent, for example as a solution in 1,3-butanediol. Among theacceptable carriers that may be employed are water, Ringer's solutionand isotonic sodium chloride solution. In some embodiments, thecomposition is formulated as a sterile, injectable solution, wherein thesolution is a sodium chloride solution (e.g., sodium chloride 0.9% USP).In some embodiments, the composition is formulated as a bolus forintradermal injection.

Examples of appropriate delivery mechanisms for intradermaladministration include, but are not limited to, implants, depots,needles, capsules, and osmotic pumps.

Methods of Treatment

Aspects of the disclosure relate to use of the compositions describedherein for treating a subject having, suspected of having or at risk ofhaving Celiac disease.

As used herein, the terms “treat”, “treating”, and “treatment” includeabrogating, inhibiting, slowing, or reversing the progression of adisease or condition, or ameliorating or preventing a clinical symptomof the disease (for example, Celiac disease). Treatment may includeinduction of immune tolerance (for example, to gluten or peptide(s)thereof), modification of the cytokine secretion profile of the subjectand/or induction of suppressor T cell subpopulations to secretecytokines. Thus, a subject treated according to the disclosurepreferably is able to eat at least wheat, rye, barley and optionallyoats without a significant T cell response which would normally lead tosymptoms of Celiac disease.

Subjects

A subject may include any subject that has or is suspected of havingCeliac disease. Preferably, the subject is a human. In some embodiments,the subject has one or more HLA-DQA and HLA-DQB susceptibility allelesencoding HLA-DQ2.5 (DQA1*05 and DQB1*02), HLA-DQ2.2 (DQA1*02 andDQB1*02) or HLA-DQ8 (DQA1*03 and DQB1*0302). In some embodiments, thesubject is HLA-DQ2.5 positive (i.e., has both susceptibility allelesDQA1*05 and DQB1*02). In some embodiments, a subject may have a familymember that has one or more HLA-DQA and HLA-DQB susceptibility allelesencoding HLA-DQ2.5 (DQA1*05 and DQB1*02), HLA-DQ2.2 (DQA1*02 andDQB1*02) or HLA-DQ8 (DQA1*03 and DQB1*0302). In some embodiments of anyone of the methods provided herein, the subject is on a gluten-freediet.

Exemplary methods for identifying subjects having or suspected of havingCeliac disease include, but are not limited to, intestinal biopsy,serology (measuring the levels of one or more antibodies present in theserum), genotyping (see, e.g., Walker-Smith J A, et al. Arch Dis Child1990), and measurement of a T cell response. Such methods may beperformed as part of any one of the methods described herein or afterany one of the methods described herein (e.g., as a companiondiagnostic), or before any one of the methods described herein (e.g., asa first-pass screen to eliminate certain subjects before use of themethods described herein, e.g., eliminating those that do not have oneor more HLA-DQA and HLA-DQB susceptibility alleles).

Detection of serum antibodies (serology) is contemplated. The presenceof such serum antibodies can be detected using methods known to those ofskill in the art, e.g., by ELISA, histology, cytology,immunofluorescence or western blotting. Such antibodies include, but arenot limited to: IgA anti-endomysial antibody (IgA EMA), IgA anti-tissuetransglutaminase 2 antibody (IgA tTG), IgA anti-deamidated gliadinpeptide antibody (IgA DGP), and IgG anti-deamidated gliadin peptideantibody (IgG DGP). Deamidated gliadin peptide-IgA (DGP-IgA) anddeamidated gliadin peptide-IgG (DGP-IgG) can be evaluated withcommercial kits (e.g. INV 708760, 704525, and 704520, INOVA Diagnostics,San Diego, Calif.).

IgA EMA: IgA endomysial antibodies bind to endomysium, the connectivetissue around smooth muscle, producing a characteristic staining patternthat is visualized by indirect immunofluorescence. The target antigenhas been identified as tissue transglutaminase (tTG or transglutaminase2). IgA endomysial antibody testing is thought to be moderatelysensitive and highly specific for untreated (active) Celiac disease.

IgA tTG: The antigen is tTG. Anti-tTG antibodies are thought to behighly sensitive and specific for the diagnosis of Celiac disease.Enzyme-linked immunosorbent assay (ELISA) tests for IgA anti-tTGantibodies are now widely available and are easier to perform, lessobserver-dependent, and less costly than the immunofluorescence assayused to detect IgA endomysial antibodies. The diagnostic accuracy of IgAanti-tTG immunoassays has been improved further by the use of human tTGin place of the nonhuman tTG preparations used in earlier immunoassaykits. Kits for IgA tTG are commercially available (INV 708760, 704525,and 704520, INOVA Diagnostics, San Diego, Calif.).

Deamidated gliadin peptide-IgA (DGP-IgA) and deamidated gliadinpeptide-IgG (DGP-IgG) are also contemplated herein and can be evaluatedwith commercial kits (INV 708760, 704525, and 704520, INOVA Diagnostics,San Diego, Calif.).

T cell response tests are also contemplated as other testing. In someembodiments, a T cell response test comprises contacting a samplecomprising a T cell with at least one gluten peptide and measuring a Tcell response in the sample. In some embodiments, a T cell response ismeasured by measuring a level of IFN-γ, where an increased level ofIFN-γ compared to a control level (e.g., a level of IFN-γ in a samplethat has not been contacted with a gluten peptide) may identify asubject as having Celiac disease. T cell response tests are known in theart (see, e.g., PCT Publication Nos.: WO/2001/025793, WO/2003/104273,WO/2005/105129, and WO/2010/060155).

Diagnostic Methods

One aspect of the disclosure relates to methods of identifying (e.g.,diagnosing) subjects, such as subjects having or suspected of havingCeliac disease.

In some embodiments, the method comprises measuring a level of at leastone circulating cytokine or chemokine in a subject that has or issuspected of having celiac disease, wherein the subject has beenadministered a composition comprising at least one gluten peptide asdescribed herein. In some embodiments, the method further comprisesassessing the likelihood the subject has Celiac disease. In someembodiments, assessing comprises comparing the level of the at least onecirculating cytokine or chemokine to a control level of the at least onecirculating cytokine or chemokine. Levels as used herein can be absoluteor relative amounts. In some embodiments, assessing comprisesdetermining the ratio of the level of the at least one circulatingcytokine or chemokine to the control level. In some embodiments, thecontrol level of the at least one circulating cytokine or chemokine is abaseline level of the circulating cytokine or chemokine. In someembodiments, the baseline level is the level of the circulating cytokineor chemokine in the subject prior to the administration of the one ormore gluten peptides. In some embodiments of any one of the methodsprovided herein, the method can further comprise the step of determininga baseline level of the circulating cytokine or chemokine in thesubject.

In some embodiments, an elevated level of the at least one circulatingcytokine or chemokine compared to a control level, such as a baselinelevel, of the at least one circulating cytokine or chemokine indicatesthat the subject has or is likely to have celiac disease. In someembodiments, a ratio greater than 1 (e.g., greater than 2, 3, 4, 5, 6,7, 8, 9, 10 or more) of the at least one circulating cytokine orchemokine to the control level, such as a baseline level, indicates thatthe subject has or is likely to have celiac disease. In some embodimentsof any one of the methods provided herein, the method further comprisesrecording whether or not the subject has or is likely to have celiacdisease based on the level or ratio.

In some embodiments, the level of the at least one circulating cytokineor chemokine is measured in a sample, e.g., a serum, plasma or urinesample, obtained from the subject. Samples are described elsewhereherein. In some embodiments, the sample is obtained from the subjectwithin 1-24 hours, such as within 1-6 hours, of administration of thecomposition. In some embodiments, the sample is obtained from thesubject within 4-6 hours of administration of the composition.

In some embodiments of any one of the methods provided herein, themethod further comprises administering the composition comprising atleast one gluten peptide as described herein to the subject, e.g., byinjection. In some embodiments, the composition is administered viaintradermal injection. In some embodiments, the composition isadministered once. In some embodiments, the composition is administeredonce via intradermal injection.

In some embodiments of any one of the methods provided herein, themethod further comprises performing other testing. Any method of othertesting as described herein is contemplated. In some embodiments, theother testing comprises a serology test, genotyping, an intestinalbiopsy, and/or a T cell response test. In some embodiments of any one ofthe methods provided herein, the method further comprises performing oneor more additional tests on the subject. In some embodiments, the methodfurther comprises contacting a sample comprising a T cell from thesubject with a gluten peptide and measuring a T cell response in thesample. In some embodiments, a T cell response is measured by measuringa level of IFN-γ, where an increased level of IFN-γ compared to acontrol level (e.g., a level of IFN-γ in a sample that has not beencontacted with a gluten peptide) may identify a subject as having Celiacdisease. In some embodiments, a level of IFN-γ at or above a cut-offlevel (e.g., at or above 7.2 pg/ml) may identify a subject as having orlikely as having Celiac disease.

In some embodiments of any one of the methods provided herein, themethod further comprising treating or suggesting a treatment if thesubject is identified as having or likely of having celiac disease. Insome embodiments of any one of the methods provided herein, the methodfurther comprises recommending a gluten-free diet and/or providinginformation in regard thereto to the subject. In some embodiments of anyone of the methods provided herein, the method further comprisesadministering a treatment, or providing information in regard thereto,to the subject. Suitable treatments are described herein. In someembodiments, the treatment is a composition comprising a gluten peptideas described herein. In some embodiments, the treatment comprises agluten-free diet.

In some embodiments, the method further comprises orally administeringor directing the subject to consume gluten prior to the measuring step.In some embodiments, the subject is orally administered or directed toconsume gluten for at least three days. In some embodiments, themeasuring step is performed six days after the last of the gluten isorally administered or consumed.

Other aspects of the disclosure relate to a method comprising:administering to a subject that has or is suspected of having Celiacdisease a first composition comprising at least one gluten peptide in anamount selected based on an HLA genotype of the subject, measuring a Tcell response to a second composition comprising at least one glutenpeptide in a sample from the subject, and assessing the likelihood thatthe subject has Celiac disease.

In some embodiments of any one of the methods provided, the firstcomposition and the second composition comprise the same gluten peptideor peptides. In some embodiments of any of the methods provided, thesample is contacted with the second composition.

In some embodiments of any one of the methods provided, the methodfurther comprises obtaining the sample from the subject.

In some embodiments of any one of the methods provided, the the subjectis orally administered or directed to consume gluten for at least threedays.

In some embodiments of any one of the methods provided, the themeasuring step is performed six days after the last of the gluten isorally administered or consumed.

In some embodiments of any one of the methods provided, IFN-gamma ismeasured. In some embodiments of any one of the methods provided, IP-10is measured.

In some embodiments of any one of the methods provided, the amount ofthe first composition, the second composition, or each of the firstcomposition and second composition, is less than 150 micrograms if thesubject has a homozygous HLA-DQ2.5 genotype. In some embodiments of anyone of the methods provided, the amount of the first composition, thesecond composition, or each of the first composition and secondcomposition, is less than 300 micrograms if the subject has a homozygousHLA-DQ2.5 genotype.

Therapeutic Efficacy Methods

One aspect of the disclosure relates to methods of assessing theefficacy of treatment of Celiac disease (e.g., responsiveness to atherapeutic gluten peptide composition). In some embodiments, the methodcomprises (a) measuring in a subject that has been administered a firstcomposition comprising at least one gluten peptide

(i) a level of at least one circulating cytokine or chemokine, and/or

(ii) a level of at least one circulating T cell; and (b) assessing theefficacy based on the measuring. The method, in some embodiments, canfurther include (c) treating the subject, or suggesting a treatment tothe subject, based on the assessing.

In some embodiments, assessing comprises comparing the level of the atleast one circulating cytokine, chemokine, or T cell to a control levelof the at least one circulating cytokine, chemokine, or T cell. Levelsas used herein can be absolute or relative amounts. In some embodiments,assessing comprises determining the ratio of the level of the at leastone circulating cytokine, chemokine, or T cell to the control level. Insome embodiments, the control level of the at least one circulatingcytokine, chemokine, or T cell is a baseline level of the circulatingcytokine, chemokine, or T cell. In some embodiments, the baseline levelis the level of the circulating cytokine, chemokine, or T cell in thesubject prior to the administration of the one or more gluten peptides.In some embodiments of any one of the methods provided herein, themethod can further comprise the step of determining a baseline level ofthe circulating cytokine, chemokine, or T cell in the subject.

In some embodiments, the assessing comprises comparing the level of theat least one circulating cytokine or chemokine, and/or the level of atleast one circulating T cell to a circulating cytokine or chemokinecontrol level, such as a baseline level, and/or a circulating T cellcontrol level, respectively. In some embodiments, the method furthercomprises recording the level(s), the result(s) of the assessing and/orthe treatment, or suggestion for treatment, based on the assessing.

In some embodiments, a ratio of about 1 of the at least one circulatingcytokine, chemokine, or T cell compared to a control level, such as abaseline level or negative control, of the at least one circulatingcytokine, chemokine, or T cell indicates that a treatment has beeneffective. In some embodiments, a ratio of greater than 1 of the atleast one circulating cytokine, chemokine, or T cell compared to acontrol level, such as a baseline level or negative control, of the atleast one circulating cytokine, chemokine, or T cell indicates that atreatment has not been effective or completely effective. In someembodiments, a ratio of greater than or about equal to 1 of the at leastone circulating cytokine, chemokine, or T cell compared to a controllevel, such as a positive control, of the at least one circulatingcytokine, chemokine, or T cell indicates that a treatment has not beeneffective or completely effective. In some embodiments, a ratio of lessthan 1 of the at least one circulating cytokine, chemokine, or T cellcompared to a control level, such as a positive control, of the at leastone circulating cytokine, chemokine, or T cell indicates that atreatment has been effective. In some embodiments, the method furthercomprises recording whether or not the treatment has been effective orcompletely effective based on the level or ratio.

In some embodiments, a level of the at least one circulating cytokine,chemokine, or T cell that is no more than two-fold above a controllevel, such as a baseline level or negative control, of the at least onecirculating cytokine, chemokine, or T cell indicates that a treatmenthas been effective. In some embodiments, a level of the at least onecirculating cytokine, chemokine, or T cell that is two-fold or moreabove a control level, such as a baseline level or negative control, ofthe at least one circulating cytokine, chemokine, or T cell indicatesthat a treatment has not been effective or completely effective. In someembodiments, a level of IL-2 and IL-8 that are each no more thantwo-fold above a control level, such as a baseline level or negativecontrol, of IL-2 and IL-8 indicates that a treatment has been effective.In some embodiments, a level of IL-2 and IL-8 that is two-fold or moreabove a control level, such as a baseline level or negative control, ofIL-2 and IL-8 indicates that a treatment has not been effective orcompletely effective.

In some embodiments, the measuring is performed on a sample obtainedfrom the subject, e.g., a serum, plasma or urine sample. Samples aredescribed herein. In some embodiments, the method further comprisesobtaining the sample from the subject. In some embodiments, the sampleis obtained from the subject within 4-6 hours of administration of thecomposition. In some embodiments, the sample is obtained from thesubject within 1-24 hours, such as within 1-6 hours, of administrationof the composition. In some embodiments, the sample is obtained from thesubject within 4-6 hours of administration of the composition.

In some embodiments, the method further comprises administering thecomposition comprising at least one gluten peptide as described hereinto the subject, e.g., by injection or oral administration. In someembodiments, the composition is administered via intradermal injection.In some embodiments, the composition is administered once. In someembodiments, the composition is administered once via intradermalinjection.

In some embodiments, treating comprises continuing with the treatment,or suggesting comprises suggesting the subject continue with thetreatment, based on the assessing. In some embodiments, treatingcomprises ceasing the treatment, or suggesting comprises suggesting thesubject cease the treatment, based on the assessing. In someembodiments, treating comprises administering a different or additionaltreatment, or the suggesting comprises suggesting the subject be treatedwith an additional or different treatment, based on the assessing.Exemplary treatments are described herein. In some embodiments, thetreatment is a composition comprising a gluten peptide as describedherein.

In some embodiments, the method further comprises orally administeringor directing the subject to consume gluten prior to the measuring step.In some embodiments, the subject is orally administered or directed toconsume gluten for at least three days. In some embodiments, themeasuring step is performed six days after the last of the gluten isorally administered or consumed.

In some embodiments, the method further comprises performing othertesting. Any method of other testing as described herein iscontemplated. In some embodiments, the other testing comprises aserology test, genotyping, an intestinal biopsy, and/or a T-cellresponse test. In some embodiments, the method further comprisescontacting a sample comprising a T cell from the subject (e.g., a wholeblood sample) with a gluten peptide and measuring a T cell response inthe sample. In some embodiments, a T cell response is measured bymeasuring a level of IFN-γ. In some embodiments, a decreased or similarlevel of IFN-γ compared to a control level (e.g., a level of IFN-γ in asample that has not been contacted with a gluten peptide) indicates thata treatment has been effective. In some embodiments, a level of IFN-γbelow a cut-off level (e.g., below 7.2 pg/ml) indicates that a treatmenthas been effective. In some embodiments, a T cell response is measuredby measuring a level of IFN-7, where an elevated level of IFN-γ comparedto a control level (e.g., a level of IFN-γ in a sample that has not beencontacted with a gluten peptide) indicates that a treatment has not beeneffective. In some embodiments, a level of IFN-γ at or above a cut-offlevel (e.g., at or above 7.2 pg/ml) indicates that a treatment has notbeen effective.

Another aspect of the disclosure relates to methods of assessingtolerance to a gluten peptide in a subject having Celiac disease. Insome embodiments, tolerance is a state of lessened responsiveness ornon-responsiveness of the immune system to a gluten peptide.

In some embodiments, the method can be any of the methods providedherein. In one embodiment, the method comprises (a) measuring in asubject that has been administered a first composition comprising atleast one gluten peptide a level of at least one circulating cytokine orchemokine; and (b) assessing the tolerance of the subject to the atleast one gluten peptide based on the measuring. In some embodiments,the subject is a subject that has previously received or is receivingtreatment for Celiac disease. In some embodiments, the treatment is acomposition comprising a gluten peptide as described herein.

In some embodiments, assessing comprises comparing the level of the atleast one circulating cytokine or chemokine to a control level of the atleast one circulating cytokine or chemokine. Levels as used herein canbe absolute or relative amounts. In some embodiments, assessingcomprises determining the ratio of the level of the at least onecirculating cytokine or chemokine to the control level. In someembodiments, the control level of the at least one circulating cytokineor chemokine is a baseline level of the circulating cytokine orchemokine. In some embodiments, the baseline level is the level of thecirculating cytokine or chemokine in the subject prior to theadministration of the one or more gluten peptides. In some embodimentsof any one of the methods provided herein, the method can furthercomprise the step of determining a baseline level of the circulatingcytokine or chemokine in the subject.

In some embodiments, the assessing comprises comparing the level of theat least one circulating cytokine or chemokine to a circulating cytokineor chemokine control level, such as a baseline level. In someembodiments, the method further comprises recording the level(s) or theresult(s) of the assessing.

In some embodiments, a ratio of about 2 or less (e.g., less than 2, lessthan 1, or less than 0.5) of the at least one circulating cytokine orchemokine compared to a control level, such as a baseline level ornegative control, of the at least one circulating cytokine or chemokineindicates that the subject has been tolerized to the gluten peptide. Insome embodiments, a ratio of greater than about 2 (e.g., at least 2, atleast 3, at least 4, at least 5, at least 6, at least 7, at least 8, atleast 9, at least 10, at least 11, at least 12, at least 13, at least14, at least 15, at least 16, at least 17, at least 18, at least 19, atleast 20, at least 25, or at least 30) of the at least one circulatingcytokine or chemokine to a control level, such as a baseline level ornegative control, of the at least one circulating cytokine or chemokineindicates that the subject has not been tolerized to the gluten peptide.In some embodiments, the method further comprises recording whether ornot the subject has been tolerized to a gluten peptide based on thelevel or ratio.

In some embodiments, the measuring is performed on a sample obtainedfrom the subject, e.g., a serum, plasma, or urine sample. Samples aredescribed herein. In some embodiments, the method further comprisesobtaining the sample from the subject. In some embodiments, the sampleis obtained from the subject within 1-24 hours, such as within 1-6hours, of administration of the composition. In some embodiments, thesample is obtained from the subject within 4-6 hours of administrationof the composition.

In some embodiments, the method further comprises administering thecomposition comprising at least one gluten peptide as described hereinto the subject, e.g., by injection or oral administration. In someembodiments, the composition is administered via intradermal injection.In some embodiments, the composition is administered once. In someembodiments, the composition is administered once via intradermalinjection.

In some embodiments, the method further comprises treating the subjector recommending a treatment to the subject based on the assessing. Insome embodiments, treating comprises continuing with the treatment, orsuggesting comprises suggesting the subject continue with the treatment,based on the assessing. In some embodiments, treating comprises ceasingthe treatment, or suggesting comprises suggesting the subject cease thetreatment, based on the assessing. In some embodiments, treatingcomprises administering a different or additional treatment, or thesuggesting comprises suggesting the subject be treated with anadditional or different treatment, based on the assessing. Exemplarytreatments are described herein. In some embodiments, the treatment is acomposition comprising a gluten peptide as described herein. In someembodiments, the treatment comprises a gluten-free diet.

In some embodiments, the method further comprises orally administeringor directing the subject to consume gluten prior to the measuring step.In some embodiments, the subject is orally administered or directed toconsume gluten for at least three days. In some embodiments, themeasuring step is performed six days after the gluten is orallyadministered or consumed.

In some embodiments, the method further comprises performing othertesting. Any method of other testing as described herein iscontemplated. In some embodiments, the other testing comprises aserology test, genotyping, an intestinal biopsy, and/or a T cellresponse test. In some embodiments, the method further comprisescontacting a sample comprising a T cell from the subject (e.g., a wholeblood sample) with a gluten peptide and measuring a T cell response inthe sample. In some embodiments, a T cell response is measured bymeasuring a level of IFN-γ. In some embodiments, a decreased or similarlevel of IFN-γ compared to a control level (e.g., a level of IFN-γ in asample that has not been contacted with a gluten peptide) may indicatethat a subject has been tolerized to the gluten peptide. In someembodiments, a level of IFN-γ below a cut-off level (e.g., below 7.2pg/ml) may indicate that a subject has been tolerized to the glutenpeptide. In some embodiments, a T cell response is measured by measuringa level of IFN-γ, where an elevated level of IFN-γ compared to a controllevel (e.g., a level of IFN-γ in a sample that has not been contactedwith a gluten peptide) may indicate that a subject has not beentolerized to the gluten peptide. In some embodiments, a level of IFN-γat or above a cut-off level (e.g., above 7.2 pg/ml) may indicate that asubject has not been tolerized to the gluten peptide.

Circulating Cytokines and Chemokines

Aspects of the disclosure relate to circulating cytokines and/orchemokines and uses thereof in a method, composition or kit describedherein. As used herein, a “circulating cytokine or chemokine” is acytokine or chemokine present in vivo in a subject, e.g., within theblood, plasma, serum, urine etc. of the subject, that may be measured ina sample obtained from the subject, e.g., in a blood (such as plasma orserum) or urine sample. The levels of such circulating cytokines orchemokines may be increased or decreased in the subject as a result ofadministration of a composition comprising a gluten peptide to thesubject, such as for a treatment of Celiac disease. Non-limitingexamples of circulating cytokines and chemokines that can be used in anyone of the methods, compositions and kits described herein include, butare not limited to, those shown in Table 2. The sequences of the genes,mRNAs, and proteins for each cytokines/chemokine can be determined byone of ordinary skill in the art using the National Center forBiotechnology Information (NCBI) gene database atwww.ncbi.nlm.nih.gov/gene.

TABLE 2 Cytokines and chemokines. Cytokine or Cytokine or NCBI ReferenceChemokine Chemokine Symbol NCBI Human Sequences Symbol (/AlternativeSymbol) Gene ID Human Protein ID(s) Chemokine (C-C MCP-1/CCL2 6347NP_002973.1 motif) ligand 2 Chemokine (C-X- IP-10/CXCL10 3627NP_001556.2 C motif) ligand 10 Interleukin 6 IL-6 3569 NP_000591.1Interleukin 8 IL-8 3576 NP_000575.1 Granulocyte G-CSF 1440 NP_000750.1,colony- NP_001171618.1, stimulating factor NP_757373.1, NP_757374.2Interleukin 2 IL-2 3558 NP_000577.2 Interleukin 1 IL-1RA 3557NP_000568.1, receptor NP_776213.1, antagonist NP_776214.1, NP_776215.1Chemokine (C-X- GRO/CXCL1 2919 NP_001502.1 C motif) ligand 1 Chemokine(C-C EOTAXIN/CCL11 6356 NP_002977.1 motif) ligand 11 Granulocyte- GM-CSF1437 NP_000749.2 macrophage colony- stimulating factor Interleukin 10IL-10 3586 NP_000563.1 Tumor necrosis TNFa 7124 NP_000585.2 factor alphaInterferon, alpha 2 IFNa2 3440 NP_000596.2 Chemokine (C-C MIP-1b/CCL46351 NP_002975.1 motif) ligand 4 Interleukin 12 IL-12P70 (heterodimerIL-12A 3592 IL-12A of IL-12A and IL-12B) IL-12B 3593 NP_000873.2 IL-12BNP_002178.2 Interleukin 1, IL-1a 3552 NP_000566.3 alpha Interleukin 17AIL-17A 3605 NP_002181.1 Epidermal growth EGF 1950 NP_001171601.1, factorNP_001171602.1, NP_001954.2 Chemokine (C-C MIP-1a/CCL3 6348 NP_002974.1motif) ligand 3 Chemokine (C- FRACTALKINE/ 6376 NP_002987.1 X3-C motif)CX3CL1 ligand 1 Interferon gamma IFNg or IFN-γ 3458 NP_000610.2 VascularVEGF 7422 NP_001020537.2, endothelial NP_001020538.2, growth factorNP_001020539.2, NP_001020540.2, NP_001020541.2, NP_001028928.1,NP_001165093.1, NP_001165094.1, NP_001165095.1, NP_001165096.1,NP_001165097.1, NP_001165098.1, NP_001165099.1, NP_001165100.1,NP_001165101.1, NP_001191313.1, NP_001191314.1, NP_001273973.1,NP_003367.4 Interleukin 9 IL-9 3578 NP_000581.1 Fibroblast growth FGF-22247 NP_001997.5 factor 2 Interleukin 1, beta IL-1b 3553 NP_000567.1Fms-related Flt-3L 2323 NP_001191431.1, tyrosine kinase 3NP_001191432.1, ligand NP_001265566.1, NP_001265567.1 Interleukin 15IL-15 3600 NP_000576.1, NP_751915.1 Lymphotoxin TNFb/LTA 4049NP_000586.2, alpha NP_001153212.1 Interleukin 12B IL-12(P40)/IL12B 3593NP_002178.2 Chemokine (C-C MCP-3/CCL7 6354 NP_006264.2 motif) ligand 7Interleukin 4 IL-4 3565 NP_000580.1, NP_758858.1 Chemokine (C-CMDC/CCL22 6367 NP_002981.2 motif) ligand 22 Interleukin 13 IL-13 3596NP_002179.2 soluble CD40 sCD40L  959 NP_000065.1 ligand TransformingTGF-a 7039 NP_001093161.1, growth factor, NP_003227.1 alpha Interleukin3 IL-3 3562 NP_000579.2 Interleukin 5 IL-5 3567 NP_000870.1 Interleukin7 IL-7 3574 NP_000871.1, NP_001186815.1, NP_001186816.1, NP_001186817.1

In some embodiments, the at least one circulating cytokine or chemokineis MCP-1, IL-6, IL-10, IL-8, or G-CSF. In some embodiments, the at leastone circulating cytokine or chemokine is IL-2, IL-8, IL-10, or MCP-1. Insome embodiments, the at least one circulating cytokine or chemokinecomprises one or more of IL-2, IL-8, IL-10, and MCP-1. In someembodiments, the at least one circulating cytokine or chemokinecomprises IL-2, IL-8, IL-10, and MCP-1. In some embodiments, the atleast one circulating cytokine or chemokine comprises IL-8, IL-10, andMCP-1. In some embodiments, the at least one circulating cytokine orchemokine comprises IL-2, IL-10, and MCP-1. In some embodiments, the atleast one circulating cytokine or chemokine comprises IL-2, IL-8, andMCP-1. In some embodiments, the at least one circulating cytokine orchemokine comprises IL-2, IL-8, and IL-10. In some embodiments, the atleast one circulating cytokine or chemokine is MCP-1, IL-6, IL-8, orG-CSF. In some embodiments, the at least one circulating cytokine orchemokine is IL-2, IL-8, or MCP-1. In some embodiments, the at least onecirculating cytokine or chemokine comprises one or more of IL-2, IL-8,and MCP-1. In some embodiments, the at least one circulating cytokine orchemokine comprises IL-8 and MCP-1. In some embodiments, the at leastone circulating cytokine or chemokine comprises IL-2 and MCP-1. In someembodiments, the at least one circulating cytokine or chemokinecomprises IL-2 and IL-8. In some embodiments, the at least onecirculating cytokine or chemokine comprises IL-2. In some embodiments,the at least one circulating cytokine or chemokine comprises IL-8. Insome embodiments, the at least one circulating cytokine or chemokinecomprises MCP-1. In some embodiments, the at least one circulatingcytokine or chemokine comprises one or more of IL-2, IP-10, and IFN-γ.In some embodiments, the at least one circulating cytokine or chemokinecomprises IL-2, IP-10, and IFN-γ.

In some embodiments, an elevated level (e.g., an elevated level ofprotein or nucleic acid (e.g., mRNA level)) of the at least onecirculating cytokine or chemokine compared to a control level of the atleast one circulating cytokine or chemokine indicates that the subjecthas or is likely to have celiac disease. In some embodiments, methodsprovided herein comprise use of the ratio of the level of the at leastone circulating cytokine or chemokine to a control level, such as abaseline level.

In some embodiments, the level of more than one circulating cytokine orchemokine is measured, e.g., the level of 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30 or more circulating cytokines or chemokines.

Assays for detecting cytokine or chemokine protein levels include, butare not limited to, immunoassays (also referred to herein asimmune-based or immuno-based assays, e.g., Western blot or enzyme-linkedimmunosorbent assay (ELISA)), Mass spectrometry, and multiplexbead-based assays. Binding partners for protein detection can bedesigned using methods known in the art and as described herein. In someembodiments, the protein binding partners, e.g., antibodies, bind to apart of or an entire amino acid sequence of at least one cytokine orchemokine, the sequence(s) being identifiable using the Genbank IDsdescribed herein. Other examples of protein detection and quantitationmethods include multiplexed immunoassays as described for example inU.S. Pat. Nos. 6,939,720 and 8,148,171, and published U.S. PatentApplication No. 2008/0255766, and protein microarrays as described forexample in published U.S. Patent Application No. 2009/0088329.

An exemplary ELISA involves at least one binding partner, e.g., anantibody or antigen-binding fragment thereof, with specificity for theat least one cytokine or chemokine. The sample with an unknown amount ofthe at least one cytokine or chemokine can be immobilized on a solidsupport (e.g., a polystyrene microtiter plate) either non-specifically(via adsorption to the surface) or specifically (via capture by anotherbinding partner specific to the same at least one cytokine, as in a“sandwich” ELISA). After the cytokine or chemokine is immobilized, thebinding partner for the at least one cytokine or chemokine can be added,forming a complex with the immobilized at least one cytokine orchemokine. The binding partner can be attached to a detectable label asdescribed herein (e.g., a fluorophore or an enzyme), or can itself bedetected by an agent that recognizes the at least one cytokine orchemokine binding partner that is attached to a detectable label asdescribed herein (e.g., a fluorophore or an enzyme). If the detectablelabel is an enzyme, a substrate for the enzyme is added, and the enzymeelicits a chromogenic or fluorescent signal by acting on the substrate.The detectable label can then be detected using an appropriate machine,e.g., a fluorimeter or spectrophotometer, or by eye.

Assays may also include a multiplex bead-based assay, such as an assaycommercially available from Luminex (see, e.g., the MAGPIX® system).Multiplex bead-based assays are known in the art.

Assays for detecting cytokine or chemokine nucleic acid, such as RNA,include, but are not limited to, Northern blot analysis, RT-PCR,sequencing technology, RNA in situ hybridization (using e.g., DNA or RNAprobes to hybridize RNA molecules present in the sample), in situ RT-PCR(e.g., as described in Nuovo G J, et al. Am J Surg Pathol. 1993, 17:683-90; Komminoth P, et al. Pathol Res Pract. 1994, 190: 1017-25), andoligonucleotide microarray (e.g., by hybridization of polynucleotidesequences derived from a sample to oligonucleotides attached to a solidsurface (e.g., a glass wafer with addressable location, such asAffymetrix microarray (Affymetrix®, Santa Clara, Calif.)). Designingnucleic acid binding partners, such as probes, is well known in the art.In some embodiments, the nucleic acid binding partners bind to a part ofor an entire nucleic acid sequence of at least one cytokine orchemokine, the sequence(s) being identifiable using the Genbank IDsdescribed herein.

Circulating T Cells

Aspects of the disclosure relate to circulating T cells and uses thereofin a method or kit described herein. As used herein, a “circulating Tcell” is a T cell present in vivo in a subject, e.g., within the bloodof the subject, that may be measured in a sample obtained from thesubject, e.g., in a blood (such as plasma or serum) sample. The levelsof such circulating T cells may be increased or decreased in the subjectas a result of administration of a composition comprising a glutenpeptide to the subject. Non-limiting examples of circulating T cellsthat can be used in the methods and kits described herein include, butare not limited to, at least one circulating T cell that recognizes atleast one gluten peptide, e.g., a gluten peptide comprised in acomposition described herein. In some embodiments, the T cellsrecognizes at least one of: (i) a first peptide comprising the aminoacid sequence PFPQPELPY (SEQ ID NO: 1) and PQPELPYPQ (SEQ ID NO: 2),(ii) a second peptide comprising the amino acid sequence PFPQPEQPF (SEQID NO: 3) and PQPEQPFPW (SEQ ID NO: 4), and (iii) a third peptidecomprising the amino acid sequence PIPEQPQPY (SEQ ID NO: 5). A T cellthat recognizes a gluten peptide is a T cell that comprises a T cellreceptor that binds to the gluten peptide and/or that binds to thegluten peptide attached to one or more Major Histocompatibility Complex(MHC) molecules. In some embodiments, the circulating T cell is a CD4⁺ Tcell. In some embodiments, the level of more than one circulating T cellis measured. The circulating T cell may be measured by direct assessmentof T cells, for example by staining with MHC-peptide multimer and flowcytometery or by functional cytokine release assays, such asinterferon-γ secretion in plasma from whole blood incubated with thecognate peptide of the T cell population of interest (e.g., a glutenpeptide described herein) or another T cell response method describedherein or otherwise known in the art.

Assays for detecting circulating T cells include, but are not limitedto, a Major Histocompatibility Complex (MHC) tetramer assay and a T cellresponse assay. Such assays are known in the art (see, e.g., John D.Altman et al. (1996). “Phenotypic Analysis of Antigen-Specific TLymphocytes.” Science 274 (5284): 94-96; Hanne Quarsten et al. (2001)“Staining of Celiac Disease-Relevant T Cells by Peptide-DQ2 Multimers.”Journal of Immunology 167(9):4861-4868; Melinda Riki et al. (2007)“Tetramer visualization of gut-homing gluten-specific T cells in theperipheral blood of celiac disease patients.” PNAS 104(8): 2831-2836). Tcell response assays are described herein and are known in the art (see,e.g., Ontiveros N, Tye-Din J A, Hardy M Y, Anderson R P. Ex vivo wholeblood secretion of interferon-γ (IFN-γ) and IFN-γ-inducible protein-10(IP-10) measured by ELISA are as sensitive as IFN-γ ELISpot for thedetection of gluten-reactive T cells in HLA-DQ2.5+ associated celiacdisease. Clin Exp Immunol. 2014; 175:305-315).

An exemplary MHC tetramer assay involves use of DQ2(DQA1*0501/DQB1*0201) MHC molecules containing a biotin. The DQ2molecules are mixed with peptides, e.g., gluten peptides, to formDQ2-peptide complexes. Tetramers may be made by conjugating theDQ2-peptide complexes with streptavidin labeled with a fluorophore. Fortetramer staining, circulating T cells are contacted with the tetramersand the tetramers bound to the circulating T cells are then detected,e.g., by flow cytometry. Secondary T cell markers may also be used inconnection with the tetramer assay, e.g., anti-CD4 antibodies, anti-CD3antibodies, and anti-CD45RA antibodies.

Samples

Samples, as used herein, refer to biological samples taken or derivedfrom a subject, e.g., a subject having or suspected of having Celiacdisease. Examples of samples include tissue samples or fluid samples. Insome embodiments, the sample is a buccal swab or a buffy coat (e.g.,isolated from anti-coagulant treated blood such as blood treated withEDTA or citrate).

General Techniques and Definitions

Unless specifically defined otherwise, all technical and scientificterms used herein shall be taken to have the same meaning as commonlyunderstood by one of ordinary skill in the art (e.g., in cell culture,molecular genetics, immunology, immunohistochemistry, protein chemistry,and biochemistry).

Unless otherwise indicated, techniques utilized in the presentdisclosure are standard procedures, well known to those skilled in theart. Such techniques are described and explained throughout theliterature in sources such as, J. Perbal, A Practical Guide to MolecularCloning, John Wiley and Sons (1984); J. Sambrook et al., MolecularCloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press(1989); T. A. Brown (editor), Essential Molecular Biology: A PracticalApproach, Volumes 1 and 2, IRL Press (2000 and 1991); D. M. Glover andB. D. Hames (editors), DNA Cloning: A Practical Approach, Volumes 1-4,IRL Press (1995 and 1996); F. M. Ausubel et al. (editors), CurrentProtocols in Molecular Biology, Greene Pub. Associates andWiley-Interscience (1988, including all updates until present); EdHarlow and David Lane (editors) Antibodies: A Laboratory Manual, ColdSpring Harbour Laboratory, (1988); and J. E. Coligan et al. (editors),Current Protocols in Immunology, John Wiley & Sons (including allupdates until present).

In any one aspect or embodiment provided herein “comprising” may bereplaced with “consisting essentially of” or “consisting of”.

Without further elaboration, it is believed that one skilled in the artcan, based on the above description, utilize the present disclosure toits fullest extent. The following specific embodiments are, therefore,to be construed as merely illustrative, and not limitative of theremainder of the disclosure in any way whatsoever. All publicationscited herein are incorporated by reference for the purposes or subjectmatter referenced herein.

EXAMPLES Example 1: Results of the Phase I Randomized, Double-Blind,Placebo-Controlled, Multiple Ascending Dose Study in Patients withCeliac Disease

3 cohorts of subjects with HLA-DQ2.5+(heterozygous or homozygous)biopsy-proven Celiac disease on a gluten-free diet for at least 1 yearwere included in the study. The first cohort (Cohort 1) contained 12subjects who were dosed with 150 mcg of a gluten peptide composition (anequimolar composition in sodium chloride 0.9% USP of 3 peptides:ELQPFPQPELPYPQPQ (SEQ ID NO: 9), EQPFPQPEQPFPWQP (SEQ ID NO: 10), andEPEQPIPEQPQPYPQQ (SEQ ID NO: 11), each peptide comprising an N-terminalpyroglutamate and C-terminal amidated amino acid) or a placebo (sodiumchloride 0.9% USP) intradermally, twice a week for 8 weeks total. Thesecond cohort (Cohort 2) contained 13 subjects who were dosed with 300mcg of the gluten peptide composition or the placebo intradermally,twice a week for 8 weeks total. The gluten peptide composition toplacebo ratio for each of Cohorts 1 and 2 were 2:1. Both Cohorts 1 and 2received an oral gluten challenge and were assessed for gamma-interferon(gIFN) release and then returned to baseline prior to starting thetreatment regimen. The third cohort (Cohort 7) contained 14 subjects whowere dosed with 150 mcg of the peptide composition or the placebointradermally, twice a week for 8 weeks total. The peptide compositionto placebo ratio for Cohort 7 was 1:1. The subjects in Cohort 7 did notundergo an oral gluten challenge or a gIFN release assay before startingthe dosage regimen.

The progress of each subject before, during and after the trial wasassessed using multiple tests including serology (tTG-IgA, DGP-IgG,DGP-IgA, and EMA-IgA), histology, and IFNg whole blood release assay,and cytokine/chemokines in plasma (measured by MAGPIX® multiplexplatform). Plasma cytokines and chemokines were measured at severaltimepoints pre and post first and last dose.

Subject disposition is summarized in Table 3. Subject demographics aresummarized in Table 4. The extent of exposure for each subject issummarized in Table 5.

TABLE 3 Subject disposition. Placebo Placebo All All Cohort 1 Cohort 2Cohort 7 (from Cohorts (from Subjects Subjects Completion (150 mg) (300mg) (150 mg) 1 and 2) Cohort 7) Dosed Screened Status (N = 8) (N = 8) (N= 7) (N = 7) (N = 7) (N = 39) (N = 67) Screened 67 (100%) Enrolled 8(100%) 10 (100%) 7 (100%) 7 (100%) 7 (100%) 39 (100%) 39 (58%) Completed8 (100%) 6 (60%) 7 (100%) 6 (86%) 7 (100%) 34 (87%) the study asrequired Completed 8 (100%) 2 (20%) 7 (100%) 5 (71%) 7 (100%) 29 (74%)study treatment per protocol (received at least 15 of 16 doses) Receivedall 7 (88%) 2 (20%) 5 (71%) 4 (57%) 6 (86%) 24 (62%) 16 doses of studytreatment Discontinued 8 (80%) 2 (29%) 10 (26%) the study prior tocompletion

TABLE 4 Subject Demographics All Cohort 1 Cohort 2 Cohort 7 Placebosubjects (150 mg) (300 mg) (150 mg) (pooled) dosed Parameter Statistic(N = 8) (N = 10) (N = 7) (N = 14) (N = 39) Age N 8 10 7 14 39 (years)Mean 52.0 50.0 42.6 39.1 45.2 SD 11.9 10.1 5.4 15.5 13.0 Median 52.552.0 45.0 34.0 47.0 Min 31 28 33 18 18 Max 66 64 47 64 66 Race Whiten(%) 8 10 7 14 39 (100%) Sex Female n(%) 7 7 5 10 29 (74%) Male n(%) 1 32 4 10 (26%) Height N 8 10 7 14 39 (cm) Mean 167.7 170.1 168.4 170.6169.5 SD 10.0 9.8 8.3 10.0 9.4 Median 168.7 167.0 173.0 170.5 169.0 Min154 158 156 156 154 Max 186 186 179 186 186 Weight N 8 10 7 14 39 (kg)Mean 70.66 85.34 74.40 66.55 73.62 SD 11.17 13.02 11.58 12.91 14.07Median 69.20 85.05 73.00 64.10 70.50 Min 60.2 66.0 58.5 48.5 48.5 Max95.1 105.5 92.5 92.3 105.5 BMI N 8 10 7 14 39 (kg/m{circumflex over( )}2) Mean 25.24 29.55 26.13 22.81 25.63 SD 4.28 4.54 2.63 3.72 4.60Median 23.91 28.91 25.23 22.64 25.23 Min 20.7 25.2 23.3 17.2 17.2 Max33.2 40.2 30.9 32.3 40.2

TABLE 5 Summary of subject exposure Number of Number of Cohort TreatmentDose level Doses Total Dose Subjects 1 peptide 150 16 2400 7 composition1 peptide 150 15 2250 1 composition 1 Placebo 0 16 0 4 2 peptide 300 164800 2 composition 2 peptide 300 5 1500 1 composition 2 peptide 300 41200 2 composition 2 peptide 300 3 900 1 composition 2 peptide 300 2 6001 composition 2 peptide 300 1 300 3 composition 2 Placebo 0 15 0 1 2Placebo 0 10 0 1 2 Placebo 0 5 0 1 7 peptide 150 16 2400 5 composition 7peptide 150 15 2250 2 composition 7 Placebo 0 16 0 6 7 Placebo 0 15 0 1

Through random distribution of the subjects, all of the subjects whowere homozygous for HLA-DQ2.5 received the gluten peptide compositiontreatment (FIG. 1). Non-homozygous HLA-DQ2.5 subjects received eitherthe gluten peptide composition treatment or placebo (FIG. 1). Aqualitative functional HLA-DQ2.5 “dose” was estimated based on thegenotype of each subject. If the subject had the DQA1*05 allele for bothcopies of the HLA-DQA gene and had the DQB1*02 allele for both copies ofthe HLA-DQB gene (i.e., was homozygous for HLA-DQ2.5), the functionalHLA-DQ2.5 dose was high. If the subject had the DQA1*05 allele for onecopy of the HLA-DQA gene and had the DQB1*02 allele for one copy of theHLA-DQB gene (i.e., was heterozygous for HLA-DQ2.5), the functionalHLA-DQ2.5 dose was low. If the subject had the HLA-DQ2.5/2.2 genotype(i.e., the subject had two DQB1*02 alleles for the HLA-DQB gene and onecopy of the DQA1*05 allele for the HLA-DQA gene), the functionalHLA-DQ2.5 dose was intermediate because the subject was homozygous forDQB1*02 and heterozygous for DQA1*05. If the subject had the HLA-DQ2.5/7genotype (i.e., the subject had one DQB1*02 allele for the HLA-DQB geneand two copies of the DQA1*05 allele for the HLA-DQA gene), thefunctional HLA-DQ2.5 dose was intermediate because the subject washeterozygous for DQB1*02 and homozygous for DQA1*05.

It was found that subjects that had a high functional HLA-DQ2.5 dose(i.e., were homozygous for HLA-DQ2.5) had generally increased levels ofcirculating cytokines in response to administration of the glutenpeptide composition when received at a dose of 150 micrograms comparedto subjects with other genotypes that received the same dose of thegluten peptide composition (FIG. 2, FIG. 3 and FIG. 4). It was alsofound that subjects that had a high functional HLA-DQ2.5 dose (i.e.,were homozygous for HLA-DQ2.5) had more adverse symptoms in response toadministration of the gluten peptide composition when received at a doseof 300 micrograms compared to subjects with other genotypes thatreceived the same dose of the gluten peptide composition (FIG. 2).

It was also found that subjects that had a high functional HLA-DQ2.5dose (i.e., were homozygous for HLA-DQ2.5) had more treatment emergentadverse events in response to administration of the gluten peptidecomposition when received at a dose of 300 micrograms or 150 microgramscompared to subjects with other genotypes that received the same dose ofthe gluten peptide composition (Table 6).

TABLE 6 Treatment emergent adverse events Total # # HLA-DQ2.5 moderate/# Subjects Total # homozygous severe AEs reporting moderate/ # HLA-DQ2.5subjects reported by moderate/ severe homozygous reporting HLA-DQ2.5 #Subjects severe AEs subjects in moderate/ homozygous in Cohort AEsreported cohort severe AEs subjects Cohort 1 8 6 11 4 4 8 Cohort 2 10 812 1 1 3 Cohort 7 7 3 4 2 2 3

As a result, it is expected that a subject who is homozygous forHLA-DQ2.5 may benefit from a lower dosage of a gluten peptide treatmentcompared to subjects who have a non-homozygous genotype (e.g., areHLA-DQ2.5 heterozygotes).

Example 2: Preparation of a 150 Microgram Dosage Composition of theFirst, Second, and Third Peptide

A dose of 150 tg the peptide composition was defined by there being 50tg (26.5 nmol) of pure peptide 1 (ELQPFPQPELPYPQPQ (SEQ ID NO: 9))comprising an N-terminal pyroglutamate and C-terminal amidated aminoacid), and an equimolar amount of peptide 2 and peptide 3(EQPFPQPEQPFPWQP (SEQ ID NO: 10)) and EPEQPIPEQPQPYPQQ (SEQ ID NO: 11)),respectively, each peptide comprising an N-terminal pyroglutamate andC-terminal amidated amino acid). The molar equivalent of 50 gg peptide 1was given by 50 gg/1889.3 g/mol=26.5 nmol. When preparing a solutioncontaining 150 gg of the peptide composition, for the constituentpeptides, the weight of each peptide was adjusted according to peptidepurity and peptide content of the lyophilized stock material. Forexample, if the peptide 1 stock material had peptide purity of 98% andits peptide content was 90%, the weight of stock material yielding 50 tgpeptide 1 was 50 gg/(peptide purity×peptide content)=50ug/(0.98×0.90)=56.7 ug.

The molar amount of peptide 1 in 150 gg of the peptide composition was26.5 nmol, and the weight of lyophilized peptide 2 stock material wastherefore given by 26.5 nmol×1833.2 g/mol/(peptide purity×peptidecontent). For example, if peptide 2 peptide purity was 99%, and peptidecontent of 95%, the mass of stock required was 51.7 ug.

The molar amount of peptide 3 in 150 ug of the peptide composition was26.5 nmol, and the weight of lyophilized peptide 3 stock material wastherefore given by 26.5 nmol×1886.2 g/mol/(peptide purity×peptidecontent). For example, if peptide 3 peptide purity was 98%, and peptidecontent of 92%, the mass of stock required was 55.4 ug.

Other Embodiments

All of the features disclosed in this specification may be combined inany combination. Each feature disclosed in this specification may bereplaced by an alternative feature serving the same, equivalent, orsimilar purpose. Thus, unless expressly stated otherwise, each featuredisclosed is only an example of a generic series of equivalent orsimilar features.

From the above description, one skilled in the art can easily ascertainthe essential characteristics of the present disclosure, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the disclosure to adapt it to various usages andconditions. Thus, other embodiments are also within the claims.

EQUIVALENTS

While several inventive embodiments have been described and illustratedherein, those of ordinary skill in the art will readily envision avariety of other means and/or structures for performing the functionand/or obtaining the results and/or one or more of the advantagesdescribed herein, and each of such variations and/or modifications isdeemed to be within the scope of the inventive embodiments describedherein. More generally, those skilled in the art will readily appreciatethat all parameters, dimensions, materials, and configurations describedherein are meant to be exemplary and that the actual parameters,dimensions, materials, and/or configurations will depend upon thespecific application or applications for which the inventive teachingsis/are used. Those skilled in the art will recognize, or be able toascertain using no more than routine experimentation, many equivalentsto the specific inventive embodiments described herein. It is,therefore, to be understood that the foregoing embodiments are presentedby way of example only and that, within the scope of the appended claimsand equivalents thereto, inventive embodiments may be practicedotherwise than as specifically described and claimed. Inventiveembodiments of the present disclosure are directed to each individualfeature, system, article, material, kit, and/or method described herein.In addition, any combination of two or more such features, systems,articles, materials, kits, and/or methods, if such features, systems,articles, materials, kits, and/or methods are not mutually inconsistent,is included within the inventive scope of the present disclosure.

All definitions, as defined and used herein, should be understood tocontrol over dictionary definitions, definitions in documentsincorporated by reference, and/or ordinary meanings of the definedterms.

All references, patents and patent applications disclosed herein areincorporated by reference with respect to the subject matter for whicheach is cited, which in some cases may encompass the entirety of thedocument.

The indefinite articles “a” and “an,” as used herein in thespecification and in the claims, unless clearly indicated to thecontrary, should be understood to mean “at least one.”

The phrase “and/or,” as used herein in the specification and in theclaims, should be understood to mean “either or both” of the elements soconjoined, i.e., elements that are conjunctively present in some casesand disjunctively present in other cases. Multiple elements listed with“and/or” should be construed in the same fashion, i.e., “one or more” ofthe elements so conjoined. Other elements may optionally be presentother than the elements specifically identified by the “and/or” clause,whether related or unrelated to those elements specifically identified.Thus, as a non-limiting example, a reference to “A and/or B”, when usedin conjunction with open-ended language such as “comprising” can refer,in one embodiment, to A only (optionally including elements other thanB); in another embodiment, to B only (optionally including elementsother than A); in yet another embodiment, to both A and B (optionallyincluding other elements); etc.

As used herein in the specification and in the claims, “or” should beunderstood to have the same meaning as “and/or” as defined above. Forexample, when separating items in a list, “or” or “and/or” shall beinterpreted as being inclusive, i.e., the inclusion of at least one, butalso including more than one, of a number or list of elements, and,optionally, additional unlisted items. Only terms clearly indicated tothe contrary, such as “only one of” or “exactly one of,” or, when usedin the claims, “consisting of,” will refer to the inclusion of exactlyone element of a number or list of elements. In general, the term “or”as used herein shall only be interpreted as indicating exclusivealternatives (i.e. “one or the other but not both”) when preceded byterms of exclusivity, such as “either,” “one of,” “only one of,” or“exactly one of.” “Consisting essentially of,” when used in the claims,shall have its ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, the phrase “atleast one,” in reference to a list of one or more elements, should beunderstood to mean at least one element selected from any one or more ofthe elements in the list of elements, but not necessarily including atleast one of each and every element specifically listed within the listof elements and not excluding any combinations of elements in the listof elements. This definition also allows that elements may optionally bepresent other than the elements specifically identified within the listof elements to which the phrase “at least one” refers, whether relatedor unrelated to those elements specifically identified. Thus, as anon-limiting example, “at least one of A and B” (or, equivalently, “atleast one of A or B,” or, equivalently “at least one of A and/or B”) canrefer, in one embodiment, to at least one, optionally including morethan one, A, with no B present (and optionally including elements otherthan B); in another embodiment, to at least one, optionally includingmore than one, B, with no A present (and optionally including elementsother than A); in yet another embodiment, to at least one, optionallyincluding more than one, A, and at least one, optionally including morethan one, B (and optionally including other elements); etc.

It should also be understood that, unless clearly indicated to thecontrary, in any methods claimed herein that include more than one stepor act, the order of the steps or acts of the method is not necessarilylimited to the order in which the steps or acts of the method arerecited.

In the claims, as well as in the specification above, all transitionalphrases such as “comprising,” “including,” “carrying,” “having,”“containing,” “involving,” “holding,” “composed of,” and the like are tobe understood to be open-ended, i.e., to mean including but not limitedto. Only the transitional phrases “consisting of” and “consistingessentially of” shall be closed or semi-closed transitional phrases,respectively, as set forth in the United States Patent Office Manual ofPatent Examining Procedures, Section 2111.03.

1. A method, comprising: selecting or adjusting a gluten peptidetreatment for a subject that has or is suspected of having Celiacdisease based on a human leukocyte antigen (HLA) genotype of thesubject.
 2. The method of claim 1, further comprising: assessing the HLAgenotype of the subject.
 3. The method of claim 2, wherein assessingcomprises determining the sequence of each copy of an HLA-DQA gene andeach copy of an HLA-DQB gene in the subject.
 4. The method of claim 3,wherein determining comprises performing a nucleic-acid based assay oneach copy of the HLA-DQA gene, or a portion thereof, and each copy ofthe HLA-DQB gene, or a portion thereof.
 5. The method of claim 4,wherein the nucleic-acid based assay is a probe-based assay or asequencing assay.
 6. The method of claim 1, wherein assessing furthercomprises identifying the subject as having a homozygous HLA-DQ2.5genotype or a non-homozygous HLA-DQ2.5 genotype.
 7. The method of claim6, wherein the non-homozygous HLA-DQ2.5 genotype is a heterozygousHLA-DQ2.5 genotype.
 8. The method of claim 7, wherein the heterozygousHLA-DQ2.5 genotype is HLA-DQ^(2.5/2.2), HLA-DQ^(2.5/7), orHLA-DQ^(2.5/8).
 9. The method of claim 1, wherein the method furthercomprises: decreasing a dose of the gluten peptide treatment if thesubject has a homozygous HLA-DQ2.5 genotype; or maintaining orincreasing the dose of the gluten peptide treatment if the subject has anon-homozygous HLA-DQ2.5 genotype.
 10. The method of claim 1, whereinthe gluten peptide treatment comprises a composition comprising: a firstpeptide comprising the amino acid sequence PFPQPELPY (SEQ ID NO: 1) andthe amino acid sequence PQPELPYPQ (SEQ ID NO:2); a second peptidecomprising the amino acid sequence PFPQPEQPF (SEQ ID NO:3) and the aminoacid sequence PQPEQPFPW (SEQ ID NO:4); and a third peptide comprisingthe amino acid sequence EQPIPEQPQ (SEQ ID NO:6) and the amino acidsequence PIPEQPQPY (SEQ ID NO:5).
 11. The method of claim 10, wherein:the first peptide comprises the amino acid sequence LQPFPQPELPYPQPQ (SEQID NO: 62); the second peptide comprises the amino acid sequenceQPFPQPEQPFPWQP (SEQ ID NO: 7); and the third peptide comprises the aminoacid sequence PEQPIPEQPQPYPQQ (SEQ ID NO: 8).
 12. The method of claim11, wherein: the first peptide comprises the amino acid sequenceELQPFPQPELPYPQPQ (SEQ ID NO: 9), wherein the N-terminal glutamate is apyroglutamate and the C-terminal glutamine is amidated; the secondpeptide comprises the amino acid sequence EQPFPQPEQPFPWQP (SEQ ID NO:10), wherein the N-terminal glutamate is a pyroglutamate and theC-terminal proline is amidated; and the third peptide comprises theamino acid sequence EPEQPIPEQPQPYPQQ (SEQ ID NO: 11), wherein theN-terminal glutamate is a pyroglutamate and the C-terminal glutamine isamidated.
 13. The method of claim 9, wherein the dose is or is decreasedto less than 300 micrograms if the subject has a homozygous HLA-DQ2.5genotype.
 14. The method of claim 13, wherein the dose is or isdecreased to less than 150 micrograms if the subject has a homozygousHLA-DQ2.5 genotype.
 15. A method, comprising: measuring a level of atleast one circulating cytokine or chemokine in a subject that has or issuspected of having Celiac disease, wherein the subject has beenadministered a first composition comprising at least one gluten peptidein an amount selected based on a human leukocyte antigen (HLA) genotypeof the subject, and assessing the likelihood the subject has Celiacdisease.
 16. The method of claim 16, further comprising: assessing theHLA genotype of the subject.
 17. The method of claim 16, whereinassessing comprises determining the sequence of each copy of an HLA-DQAgene and each copy of an HLA-DQB gene in the subject.
 18. The method ofclaim 17, wherein determining comprises performing a nucleic-acid basedassay on each copy of the HLA-DQA gene, or a portion thereof, and eachcopy of the HLA-DQB gene, or a portion thereof. 19-46. (canceled)
 47. Amethod for assessing tolerance to a gluten peptide in a subject havingCeliac disease, the method comprising: measuring a level of at least onecirculating cytokine or chemokine in a subject having Celiac disease,wherein the subject has been administered a first composition comprisingat least one gluten peptide in an amount selected based on a humanleukocyte antigen (HLA) genotype of the subject, and assessing thetolerance of the subject to the at least one gluten peptide based on themeasuring. 48-78. (canceled)
 79. A method comprising: administering to asubject that has or is suspected of having Celiac disease a firstcomposition comprising at least one gluten peptide in an amount selectedbased on an HLA genotype of the subject, measuring a T cell response toa second composition comprising at least one gluten peptide in a samplefrom the subject, and assessing the likelihood that the subject hasCeliac disease. 80-87. (canceled)